Anti-IGF-1r antibody compositions

Abstract

The present disclosure relates generally to pharmaceutical compositions comprising an anti-IGF-1R antibody and methods of making and using such compositions. In certain embodiments, such compositions exhibit low viscosity and high stability, and thus can be conveniently delivered to subjects in need thereof while reducing discomfort. Also provided herein are anti-IGF-1R antibodies having a heavy chain comprising a charged amino acid located on the c-terminus thereof.

Description

Background Art

[0001] Thyroid eye disease (TED) (also known as Graves' ophthalmopathy) is an autoimmune inflammatory disorder of the orbit and periorbital tissues characterized by upper eyelid retraction, eyelid lag, swelling, redness (erythema), conjunctivitis, and proptosis (exophthalmos). It is most common in individuals with Graves' disease and less common in individuals with Hashimoto's thyroiditis or individuals with normal thyroid function. It is part of a systemic process, expressed differently in the eye, thyroid, and skin, and is caused by autoantibodies that bind to tissues in these organs. These autoantibodies target fibroblasts in the eye muscles, and these fibroblasts can differentiate into fat cells (fat cells / adipocytes). The fat cells and muscles dilate and become inflamed. Veins are compressed and unable to drain fluid, resulting in edema.

[0002] Current treatments for hyperthyroidism due to Graves' disease are incomplete due to the lack of therapies that target the specific underlying pathogenic autoimmune mechanisms of the disease. Even more complicated is the treatment of moderate to severe active TED. Although recent years have witnessed a better understanding of its pathogenesis, TED still presents treatment challenges and dilemmas. Therefore, there is a significant unmet need for the treatment of patients suffering from TED and its associated symptoms. Summary of the invention

[0003] There is an unmet need to develop therapies for treating TED with greater depth and durability of response. In addition, currently available therapies for treating TED have side effects, including hearing impairment. These side effects, which are reported in a significant proportion of patients receiving currently available therapies, have limited treatment in patients with TED.

[0004] In certain aspects, provided herein are pharmaceutical compositions comprising anti-IGF-1R antibodies and methods of using and preparing such pharmaceutical compositions. In certain embodiments, the pharmaceutical compositions of the present invention are IGF-1R antibody solutions. In certain embodiments, the pharmaceutical compositions provided herein comprise high concentrations of anti-IGF-1R antibody solutions (e.g., greater than 75 mg / ml or greater than 100 mg / ml), while exhibiting high pharmaceutical stability and low viscosity. Therefore, such compositions are suitable for subcutaneous delivery (including self-delivery) to patients with thyroid eye disease and other diseases and conditions for which anti-IGF-1R antibodies can provide therapeutic benefits. In addition, the high antibody concentration of the compositions provided herein coupled with low viscosity allows for delivery of lower volumes of compositions to patients through smaller needles, thereby reducing patient discomfort and further promoting self-delivery.

[0005] In certain aspects, provided herein is a pharmaceutical composition comprising: (a) at least 75 mg / ml anti-IGF-1R antibody; (b) 20 mM to 30 mM histidine; and (c) 4% to 6% sugar; wherein the pH range of the pharmaceutical composition is 5.5-6.5. In some embodiments, the sugar is D-sorbitol.

[0006] In certain embodiments, the anti-IGF-1R antibody comprises CDRH1 of SEQ ID NO: 1, CDRH2 of SEQ ID NO: 2, CDRH3 of SEQ ID NO: 3, CDRL1 of SEQ ID NO: 4, CDRL2 of SEQ ID NO: 5, and CDRL3 of SEQ ID NO: 6. In some embodiments, the antibody comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 7. In some embodiments, the antibody comprises a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 8. In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, the antibody comprises a heavy chain further comprising a charged amino acid located at its c-terminus (e.g., a c-terminal arginine, histidine, lysine, aspartic acid, or glutamic acid). In some embodiments, the charged amino acid is a positively charged amino acid (e.g., arginine, histidine, or lysine). In some embodiments, the charged amino acid is a negatively charged amino acid (e.g., aspartic acid or glutamic acid). In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 11, 12, 13, 14 or 15. In some embodiments, the antibody comprises a human IgG1 heavy chain constant domain and a human kappa light chain constant domain. In certain embodiments, the anti-IGF-1R antibody is lonigutamab.

[0007] Ronituzumab is a humanized monoclonal antibody against IGF-1R. Ronituzumab has high affinity and specificity for IGF-1R. Specifically, Ronituzumab has picomolar affinity for IGF-1R. In certain preferred embodiments, the k of the binding epitope of Ronituzumab to IGF-1R is DThe therapeutic effect of ronitumomab can be obtained within about one hour after the pharmaceutical composition comprising ronitumomab is administered to the patient.

[0008] Ronituzumab has unique and beneficial pharmacological properties. In certain preferred embodiments, administration of a pharmaceutical composition comprising Ronituzumab induces greater than about 70% IGF-1R internalization. In certain preferred embodiments, administration of a pharmaceutical composition comprising Ronituzumab induces greater than about 80% IGF-1R internalization. In certain preferred embodiments, administration of a pharmaceutical composition comprising Ronituzumab induces greater than about 85% IGF-1R internalization. In certain preferred embodiments, administration of a pharmaceutical composition comprising Ronituzumab induces greater than about 90% IGF-1R internalization.

[0009] In certain embodiments, the pharmaceutical composition comprises at least 75 mg / ml of the anti-IGF-1R antibody. In some embodiments, the pharmaceutical composition comprises at least 100 mg / ml, at least 125 mg / ml, at least 150 mg / ml, at least 175 mg / ml, at least 200 mg / ml, or at least 250 mg / ml of the anti-IGF-1R antibody. In certain embodiments, the pharmaceutical composition comprises 75 mg / ml to 300 mg / ml, 100 mg / ml to 300 mg / ml, or 125 mg / ml to 250 mg / ml of the anti-IGF-1R antibody. In some embodiments, the pharmaceutical composition comprises about 125 mg / ml, about 150 mg / ml, about 175 mg / ml, about 200 mg / ml, or about 250 mg / ml of the anti-IGF-1R antibody.

[0010] In some embodiments, the pharmaceutical composition comprises 20mM to 30mM histidine. In certain embodiments, the pharmaceutical composition comprises about 20mM, about 21mM, about 22mM, about 23mM, about 24mM, about 25mM, about 26mM, about 27mM, about 28mM, about 29mM or about 30mM histidine.

[0011] In some embodiments, the pharmaceutical composition comprises 4% to 6% sugar (eg, D-sorbitol). In certain embodiments, the pharmaceutical composition comprises about 4%, 5%, or 6% sugar (eg, D-sorbitol).

[0012] In some embodiments, the pH of the pharmaceutical composition is 5.5 to 6.5. In certain embodiments, the pH of the pharmaceutical composition is about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, or about 6.5.

[0013] In some embodiments, the pharmaceutical composition does not contain polysorbate 80. In some embodiments, the pharmaceutical composition contains a small amount of polysorbate 80 (e.g., less than 0.05%). In certain embodiments, the pharmaceutical composition contains no more than 0.05% polysorbate 80. In some embodiments, the pharmaceutical composition contains no more than 0.02% polysorbate 80. In some embodiments, the pharmaceutical composition contains no more than 0.01% polysorbate 80. In some embodiments, the pharmaceutical composition contains no more than 0.005% polysorbate 80. In certain embodiments, the pharmaceutical composition contains 0.002% to 0.05% polysorbate 80. In some embodiments, the pharmaceutical composition comprises about 0.002%, about 0.003%, about 0.004%, about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.015%, about 0.02%, about 0.025%, about 0.03%, about 0.035%, about 0.04%, about 0.045%, or about 0.05% polysorbate 80.

[0014] In some embodiments, the pharmaceutical composition does not contain poloxamer 188 (P188). In some embodiments, the pharmaceutical composition contains a small amount of poloxamer 188 (e.g., less than 0.1%). In certain embodiments, the pharmaceutical composition contains no more than 0.1% poloxamer 188. In some embodiments, the pharmaceutical composition contains no more than 0.05% poloxamer 188. In some embodiments, the pharmaceutical composition contains no more than 0.02% poloxamer 188. In some embodiments, the pharmaceutical composition contains no more than 0.01% poloxamer 188. In some embodiments, the pharmaceutical composition further contains 0.01% to 0.1% poloxamer 188. In certain embodiments, the pharmaceutical composition contains about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09% or about 0.1% poloxamer 188.

[0015] In certain embodiments, the osmotic pressure of the pharmaceutical composition is within the physiological osmotic pressure range of 250-400 mOsm / kg.

[0016] In certain embodiments, the viscosity of the pharmaceutical composition is no more than 30 cP at 21° C. In certain embodiments, the viscosity of the pharmaceutical composition is no more than 15 cP at 21° C. In certain embodiments, the viscosity of the pharmaceutical composition is about 10 cP, about 11 cP, about 12 cP, about 13 cP, about 14 cP, about 15 cP, about 16 cP, about 17 cP, about 18 cP, about 19 cP, about 20 cP, about 21 cP, about 22 cP, about 23 cP, about 24 cP, about 25 cP, about 26 cP, about 27 cP, about 28 cP, about 29 cP, or about 30 cP at 21° C.

[0017] In certain embodiments, the pharmaceutical composition is stable for at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, at least 13 weeks, at least 14 weeks, at least 15 weeks, at least 16 weeks, or longer. In some embodiments, the pharmaceutical composition is stable at a temperature of -70°C to 8°C (e.g., about -70°C, about -20°C, about 4°C) for this length of time.

[0018] In some aspects, a kind of syringe (injector or syringe) is provided herein, and it comprises the pharmaceutical composition provided herein.In some embodiments, the syringe comprises the delivery volume of no more than 2ml (for example, no more than 1.5ml, no more than 1ml).In some embodiments, the syringe comprises the needle of size no more than 24G (for example, 25G, 26G).In certain embodiments, the syringe is an automated reusable fixed dose device.In some embodiments, the syringe is an automated reusable variable dose device.In some embodiments, the syringe is an automated disposable fixed dose automatic syringe.

[0019] In certain aspects, provided herein is an anti-IGF-1R antibody comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, a CDRH3 of SEQ ID NO: 3, a CDRL1 of SEQ ID NO: 4, a CDRL2 of SEQ ID NO: 5, and a CDRL3 of SEQ ID NO: 6, wherein the heavy chain of the antibody comprises a charged amino acid located at its c-terminus (e.g., a c-terminal arginine, histidine, lysine, aspartic acid, or glutamic acid). In some embodiments, the charged amino acid is a positively charged amino acid (e.g., arginine, histidine, or lysine). In some embodiments, the charged amino acid is a negatively charged amino acid (e.g., aspartic acid or glutamic acid). In some embodiments, the antibody comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 7. In some embodiments, the antibody comprises a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 11, 12, 13, 14, or 15. In some embodiments, provided herein is a pharmaceutical composition comprising an anti-IGF-1R antibody.

[0020] In certain aspects, provided herein are methods of treating a disease or condition comprising administering a pharmaceutical composition and / or antibody as provided herein. In some embodiments, the pharmaceutical composition is administered using a syringe as provided herein. In certain embodiments, the disease or condition is thyroid eye disease (TED). In some embodiments, the disease or disorder is stroke, acromegaly, diabetic nephropathy / diabetic kidney disease, idiopathic pulmonary fibrosis, interstitial lung disease, obesity, type 2 diabetes, juvenile idiopathic arthritis (JIA), diffuse cutaneous systemic sclerosis, Sjogren's syndrome calcification and vasculitis, cachexia and sarcopenia, diabetic macular edema, atherosclerosis, peripheral arterial disease (PAD), myocardial infarction and stroke, diabetic foot and skin, rheumatoid arthritis, neurofibromatosis 1, neurofibromatosis 2, polycystic kidney disease, polycystic liver disease, polycystic ovary syndrome, Alzheimer's disease, cognitive decline, dementia, depression and anxiety, asthma, aging, thyroid eye disease, idiopathic orbital inflammation, human type 2 lipodystrophy and related cardiomyopathy, autosomal dominant polycystic kidney disease (ADPKD), NASH, Graves disease (Graves disease), disease) and / or Hashimoto's thyroiditis.

[0021] In certain embodiments, the pharmaceutical compositions and / or antibodies provided herein are used to treat a disease or condition. In some embodiments, the disease or condition is thyroid eye disease (TED). In some embodiments, the disease or disorder is stroke, acromegaly, diabetic nephropathy (diabetic kidney disease), idiopathic pulmonary fibrosis, interstitial lung disease, obesity, type 2 diabetes, juvenile idiopathic arthritis (JIA), diffuse cutaneous systemic sclerosis, Sjögren's syndrome calcification and vasculitis, cachexia and sarcopenia, diabetic macular edema, atherosclerosis, peripheral arterial disease (PAD), myocardial infarction and stroke, diabetic foot and skin, rheumatoid arthritis, neurofibromatosis 1, neurofibromatosis 2, polycystic kidney disease, polycystic liver disease, polycystic ovary syndrome, Alzheimer's disease, cognitive decline, dementia, depression and anxiety, asthma, aging, thyroid eye disease, idiopathic orbital inflammation, human type 2 lipodystrophy and related cardiomyopathy, autosomal dominant polycystic kidney disease (ADPKD), NASH, Graves' disease and / or Hashimoto's thyroiditis.

[0022] In certain embodiments of the compositions and methods provided herein, the pharmaceutical composition is formulated for subcutaneous administration. In certain embodiments, the pharmaceutical composition is administered subcutaneously. In certain embodiments, the pharmaceutical composition is formulated for intramuscular administration. In certain embodiments, the pharmaceutical composition is administered intramuscularly. In certain embodiments, the pharmaceutical composition is formulated for suborbital, intravitreal, intraocular, subconjunctival, retrobulbar, periorbital and / or intrathecal administration. In certain embodiments, the pharmaceutical composition is administered by suborbital, intravitreal, intraocular, subconjunctival, retrobulbar, periorbital and / or intrathecal injection.

[0023] In certain embodiments, the pharmaceutical composition is administered in a delivery volume of no more than 3 ml, 2.5 ml, 2 ml, 1.5 ml, or 1 ml. In some embodiments, the pharmaceutical composition is administered in a delivery volume of no more than 2 ml.

[0024] In certain embodiments, the pharmaceutical composition is administered through a needle no larger than 24G, 25G, or 27G in size. In some embodiments, the pharmaceutical composition is administered in a 24G needle, a 25G needle, or a 27G needle.

[0025] In certain embodiments, the pharmaceutical composition is administered with an injection force of no more than 14 N, 13 N, 12 N, 11 N, 10 N, 9 N, 8 N, 7 N, or 6 N. In certain embodiments, the pharmaceutical composition is administered with an injection force of no more than 12 N. In certain embodiments, the pharmaceutical composition is administered with an injection force of about 4 N, about 5 N, about 6 N, about 7 N, about 8 N, about 9 N, about 10 N, about 11 N, or about 12 N.

[0026] In certain embodiments, the methods provided herein treat and / or prevent one or more diseases or conditions. In some embodiments, the disease or condition is thyroid eye disease (TED). In some embodiments, the disease or disorder is stroke, acromegaly, diabetic nephropathy (diabetic kidney disease), idiopathic pulmonary fibrosis, interstitial lung disease, obesity, type 2 diabetes, juvenile idiopathic arthritis (JIA), diffuse cutaneous systemic sclerosis, Sjögren's syndrome calcification and vasculitis, cachexia and sarcopenia, diabetic macular edema, atherosclerosis, peripheral arterial disease (PAD), myocardial infarction and stroke, diabetic foot and skin, rheumatoid arthritis, neurofibromatosis 1, neurofibromatosis 2, polycystic kidney disease, polycystic liver disease, polycystic ovary syndrome, Alzheimer's disease, cognitive decline, dementia, depression and anxiety, asthma, aging, thyroid eye disease, idiopathic orbital inflammation, human type 2 lipodystrophy and related cardiomyopathy, autosomal dominant polycystic kidney disease (ADPKD), NASH, Graves' disease and / or Hashimoto's thyroiditis.

[0027] In certain embodiments, the method reduces the severity of the thyroid eye disease (TED). In the method, proptosis in an eye of a subject with thyroid eye disease (TED) is reduced. In some embodiments, proptosis is reduced by at least 2 mm, at least 3 mm, or at least 4 mm.

[0028] In some embodiments, the methods provided herein reduce the clinical activity score (CAS) of thyroid eye disease (TED). In some embodiments, the clinical activity score (CAS) is reduced by at least 2 points. In certain embodiments, the clinical activity score (CAS) is reduced to one (1). In certain embodiments, the clinical activity score (CAS) of the subject is reduced to zero (0).

[0029] In some embodiments, the methods provided herein improve the subject's European Graves' Orbitopathy Group Clinical Activity Score (EUGOGO CAS) (e.g., provides at least 1, 2, or 3). In certain embodiments, the method improves the Clinical Severity Scale Score (CMSS) of pa (e.g., improves at least 1, 2, or 3).

[0030] In certain embodiments, the method provided herein improves the quality of life of the experimenter. In certain embodiments, the quality of life is measured by Graves' Ophthalmopathy Quality of Life (GO-QoL) assessment. In certain embodiments, the quality of life is measured by its visual function or appearance subscale. In certain embodiments, the quality of life is measured by the European Graves' Orbitopathy Group (EUGOGO) guide. In certain embodiments, the method provided herein reduces the severity of diplopia (for example, persistent diplopia, non-persistent diplopia, intermittent diplopia).

[0031] In certain aspects, the present invention provides methods of administering pharmaceutical compositions comprising ronituzumab. These pharmaceutical compositions can be administered to patients suffering from thyroid eye disease (TED). In certain embodiments, the pharmaceutical compositions of the present invention can be administered intravenously. The methods of the present invention provide for intravenous infusion of a pharmaceutical composition comprising up to about 3.0 mg / kg ronituzumab to the patient. In certain embodiments, the methods of the present invention provide for administering a pharmaceutical composition comprising about 0.1 mg / kg, 0.3 mg / kg, 1.0 mg / kg or 3.0 mg / kg ronituzumab in the form of an intravenous infusion. The present invention further provides that the intravenous infusion can occur for a period of about 15 minutes to about 120 minutes. In certain preferred embodiments, the intravenous infusion occurs for a duration of about 60 minutes.

[0032] In certain aspects, the present invention provides a pharmaceutical composition comprising ronitumumab administered in the form of a subcutaneous injection. In certain embodiments, the method of the present invention provides subcutaneous administration of up to about 250 mg ronitumumab. In certain embodiments, the pharmaceutical composition for subcutaneous administration comprises about 20 mg, 40 mg, 125 mg or 250 mg ronitumumab. The volume of the pharmaceutical composition for subcutaneous administration can be about 0.1 mL to about 3 mL. In certain embodiments, the volume of the pharmaceutical composition for subcutaneous administration can be about 0.5 mL to about 3 mL. In a preferred embodiment, the volume administered is up to about 2 mL. Beneficially, since subcutaneous administration is performed within a tolerable volume, the pharmaceutical composition can be administered in an outpatient setting, or the pharmaceutical composition of the present invention can be administered by the patient himself. This creates a convenient and efficient way to administer a pharmaceutical composition comprising ronitumumab. Specifically, compared to intravenous administration, subcutaneous administration of a pharmaceutical composition is beneficial due to the ease of administering the pharmaceutical composition. In addition, as demonstrated by the pharmacokinetic data provided herein, subcutaneous administration of a pharmaceutical composition comprising ronitumumab results in favorable pharmacokinetic and pharmacodynamic properties of ronitumumab. Specifically, subcutaneous administration of a pharmaceutical composition comprising ronitumumab results in higher and longer-lasting therapeutically effective serum concentrations of ronitumumab.

[0033] In certain embodiments of the invention, administration of a pharmaceutical composition comprising ronitumumab maximizes the IGF-1R receptor occupancy of an anti-IGF-1R antibody. This maximum IGF-1R occupancy can be achieved at any time after administration of a pharmaceutical composition comprising ronitumumab. In certain embodiments, this is achieved after about 12 hours after administration of a pharmaceutical composition comprising ronitumumab. In certain embodiments, the administered pharmaceutical composition may comprise from about 10 mg to about 500 mg ronitumumab. In certain embodiments, the administered pharmaceutical composition may comprise from about 20 mg to about 400 mg ronitumumab. In preferred embodiments, the administered pharmaceutical composition may comprise from about 100 mg to about 300 mg ronitumumab. In preferred embodiments, the administered pharmaceutical composition may comprise from about 20 mg or about 40 mg ronitumumab. In preferred embodiments, the pharmaceutical composition may comprise from about 125 mg or about 250 mg ronitumumab. It is noteworthy that the maximum IGF-1R receptor occupancy can be maintained for up to several months after a single administration. In preferred embodiments, IGF-1R receptor occupancy is maintained for at least about 4 weeks after a single administration of the pharmaceutical composition comprising ronitumumab.Maximum receptor occupancy levels may be maintained after a single subcutaneous injection or intravenous infusion of the pharmaceutical composition comprising ronitumumab.

[0034] In certain aspects, the serum concentration required for maximum IGF-1R receptor internalization is a concentration of at least about 3 μg / mL of ronituzumab. Thus, the therapeutically effective serum concentration of ronituzumab is at least about 1 μg / mL. In certain preferred embodiments, the therapeutically effective serum concentration of ronituzumab is at least about 3 μg / mL. Importantly, the pharmaceutical composition of the present invention provides a therapeutically effective serum concentration of ronituzumab to the patient.

[0035] In certain aspects of the invention, the pharmaceutical composition comprising ronitumumab administered to the patient is safe and tolerable. In addition, the pharmaceutical composition of the invention has minimal adverse events.

[0036] In certain aspects, the present invention provides methods of treating thyroid eye disease (TED), comprising subcutaneously administering a pharmaceutical composition comprising about 10 mg to about 500 mg of ronituzumab. In certain embodiments, the pharmaceutical composition comprises about 20 mg of ronituzumab. In certain embodiments, the pharmaceutical composition comprises about 40 mg of ronituzumab. In certain embodiments, the pharmaceutical composition comprises about 125 mg of ronituzumab. In certain embodiments, the pharmaceutical composition comprises about 250 mg of ronituzumab.

[0037] In certain embodiments, the pharmaceutical composition comprising ronituzumab is administered once a week. In certain embodiments, the pharmaceutical composition comprising ronituzumab is administered twice a week. In certain embodiments, the pharmaceutical composition comprising ronituzumab is administered three times a week. In certain embodiments, the pharmaceutical composition comprising ronituzumab is administered every other day (i.e., once every two days). In certain preferred embodiments, the pharmaceutical composition comprising ronituzumab is administered once every two (2) weeks. In certain preferred embodiments, the pharmaceutical composition comprising ronituzumab is administered once every three (3) weeks. In certain preferred embodiments, the pharmaceutical composition comprising ronituzumab is administered once every four (4) weeks. In certain embodiments, the pharmaceutical composition comprising ronituzumab is administered once every five (5) weeks. In certain embodiments, the pharmaceutical composition comprising ronituzumab is administered once every six (6) weeks.

[0038] In certain preferred embodiments, the pharmaceutical composition comprising ronituzumab is administered on day 1 and day 14. In other preferred embodiments, the pharmaceutical composition comprising ronituzumab is administered on day 1 and day 21. In other preferred embodiments, the pharmaceutical composition comprising ronituzumab is administered on day 1 and day 28.

[0039] In certain preferred embodiments, the pharmaceutical composition comprising ronituzumab is administered subcutaneously on day 1 and day 14. In other preferred embodiments, the pharmaceutical composition comprising ronituzumab is administered subcutaneously on day 1 and day 21. In other preferred embodiments, the pharmaceutical composition comprising ronituzumab is administered subcutaneously on day 1 and day 28. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The response surface of multimer rate after stress (histidine) is shown.

[0041] Figure 2 The response surface for increasing acid variant rates after stress (histidine) is shown.

[0042] Figure 3 Shows T m1 (histidine) response surface.

[0043] Figure 4 Shows T m2 (histidine) response surface.

[0044] Figure 5 The response surface of multimer rate after stress (phosphate) is shown.

[0045] Figure 6 The response surface for increasing acid mutation rate after stress (phosphate) is shown.

[0046] Figure 7 Thermal stability: -20°C DLS results are shown.

[0047] Figure 8 Thermal stability: 2-8°C DLS results are shown.

[0048] Fig. 9 A process flow diagram for an antibody drug product is shown.

[0049] Fig.10 A TFF process flow chart is shown.

[0050] Fig.11 Representative diagrams depicting infusion bags and ports are shown.

[0051] Fig.12 Shown is the trend of monomer content at room temperature in an in-use compatibility study.

[0052] Fig.13 The trend of the main form content at room temperature in the in-use compatibility study is shown.

[0053] Fig.14 Shown are the trends in monomer content at 2-8°C in the in-use compatibility study.

[0054] Fig.15 The trend of the major form content at 2-8°C in the in-use compatibility study is shown.

[0055] Fig.16 Data related to adverse events following administration of a pharmaceutical composition comprising ronitumumab are presented.

[0056] Fig.17 Shown are serum concentrations of ronituzumab following intravenous infusion of 0.1 mg / kg, 0.3 mg / kg, 1.0 mg / kg, and 3.0 mg / kg of ronituzumab.

[0057] Fig.18 Shown are serum concentrations of ronitumumab following subcutaneous administration of 20 mg, 40 mg, 125 mg, and 250 mg of ronitumumab.

[0058] Fig.19 Receptor occupancy-time profiles following intravenous infusion of 0.1 mg / kg, 0.3 mg / kg, 1.0 mg / kg, and 3.0 mg / kg of ronitumumab are shown.

[0059] Fig. 20 Receptor occupancy-time profiles following subcutaneous administration of 20 mg, 40 mg, 125 mg, and 250 mg of ronitumumab are shown. DETAILED DESCRIPTION

[0060] Overview

[0061] Provided herein are novel pharmaceutical compositions comprising antibodies (specifically monoclonal antibodies) capable of binding to IGF-1R and methods for preparing and using such pharmaceutical compositions. From one aspect, provided herein is a novel pharmaceutical composition comprising an antibody or antigen-binding fragment thereof capable of binding to IGF-1R and being internalized into a cell by inducing internalization of IGF-1R. Also provided herein are certain anti-IGF-1R antibodies comprising a positively charged amino acid (e.g., a C-terminal arginine, histidine, or lysine) located at its c-terminus. Also provided herein are uses of the pharmaceutical compositions and / or antibodies for preventing diseases associated with IGF-1R (e.g., thyroid eye disease), reducing the risk of developing the disease, or treating the disease.

[0062] Certain aspects of the present disclosure relate to pharmaceutical compositions and methods for preventing, reducing the risk of developing, or treating thyroid eye disease. Thyroid eye disease is a disease in which the eye muscles, eyelids, tear glands, and fatty tissue behind the eye become inflamed. This may cause redness, swelling, and discomfort in the eye and eyelids, and the eye may be thrust forward ("bulging" or "protruding" of the eye). In some cases, there is swelling and stiffness in the muscles that move the eye so that they no longer move in unison with each other; this can cause double vision. Rarely, TED may result in decreased vision due to pressure on the nerves at the back of the eye or an ulcer that forms in front of the eye if the eyelid cannot close completely.

[0063] TED, also known as Graves' orbitopathy or eye disease, is an autoimmune condition. It occurs when the body's immune system attacks the tissues around the eye, causing inflammation of the tissues around and behind the eye. In most patients, the same autoimmune condition that causes TED also affects the thyroid gland, causing Graves' disease. Graves' disease most often causes an overactive thyroid gland (hyperthyroidism), but can rarely cause an underactive thyroid gland (hypothyroidism). TED can occur in people whose thyroid glands are overactive, underactive, or functioning normally. It can also occur after treatment for Graves' disease. People with TED need to be cared for by an eye specialist (ophthalmologist) and a thyroid specialist (endocrinologist).

[0064] Currently available therapies for the treatment of TED have side effects, including hearing loss. These side effects, reported in a significant proportion of patients receiving currently available therapies, have limited viable therapies for treating patients with TED. Therefore, there is an unmet need to develop therapies for the treatment of TED with greater depth and durability of response.

[0065] definition

[0066] As used in this specification, "a" or "an" may mean one or more. As used in the claims, when used in conjunction with the word "comprising", the word "a" or "an" may mean one or more than one. For example, a reference to "antibody" is a reference from one antibody to many antibodies. As used herein, "another" may mean at least a second or more.

[0067] The term "immunoglobulin" (Ig) is used interchangeably with "antibody" herein. The term "antibody" herein is used in the broadest sense and specifically encompasses monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two complete antibodies, antibody fragments (as long as they exhibit biological activity), and antibody derivatives. The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. H and V L The pairing forms a single antigen binding site together. For the structure and characteristics of different classes of antibodies, see, for example, "Basic and Clinical Immunology", 8th edition, Daniel P.Stites, Abba I.Terr and Tristram G.Parslow (editor), Appleton & Lange, Norwalk, CT, 1994, page 71 and chapter 6. The L chain of any vertebrate species can be assigned to one of two significantly different types (referred to as κ ("kappa") and λ ("lambda")) based on the amino acid sequence of its constant domain. According to the amino acid sequence of the constant domain of its heavy chain (CH), immunoglobulin can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, which have heavy chains designated as α ("alpha"), δ ("delta"), ε ("epsilon"), γ ("gamma"), and μ ("mu"), respectively. The γ and α classes are further divided into subclasses (isotypes) based on relatively minor differences in CH sequence and function, e.g., humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and generally described in, e.g., Abbas et al., Cellular and Molecular Immunology, 4th edition (WB Saunders Co., 2000).

[0068] The "variable region" or "variable domain" of an antibody refers to the amino terminal domain of the heavy chain or light chain of an antibody. The variable domains of the heavy chain and light chain can be referred to as "V H ” and “V L". These domains are usually the most variable parts of an antibody (relative to other antibodies of the same class) and contain the antigen binding site. The variable domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the span of the variable domain. Instead, it is concentrated in three segments called hypervariable regions (HVRs) in both the light chain variable domain and the heavy chain variable domain. The more highly conserved parts of the variable domain are called framework regions (FRs). The variable domains of native heavy and light chains each contain four FR regions that largely adopt a β-sheet configuration, connected by three HVRs that form loops that connect the β-sheet structure and in some cases form a part of the β-sheet structure. The HVRs on each chain are held together in close proximity by the FR regions and, together with the HVRs from the other chain, contribute to the formation of the antigen binding site of the antibody (see Kabat et al., Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Maryland). Health, Bethesda, MD) (1991)). The constant domains are not directly involved in the binding of an antibody to an antigen, but exhibit various effector functions, such as enabling the antibody to participate in antibody-dependent cellular toxicity.

[0069] As used herein, the term "CDR" or "complementarity determining region" is intended to mean a non-contiguous antigen binding site present in the variable region of both a heavy chain polypeptide and a light chain polypeptide. CDRs have been described in Kabat et al., J. Biol. Chem. 252: 6609-6616 (1977); Kabat et al., U.S. Department of Health and Human Services, Sequences of proteins of immunological interest (1991) (also referred to herein as Kabat 1991); Chothia et al., J. Mol. Biol. 196: 901-917 (1987) (also referred to herein as Chothia 1987); and MacCallum et al., J. Mol. Biol. 262: 732-745 (1996), wherein the definition includes amino acid residues or subsets of amino acid residues that overlap when compared to each other. However, the application of any definition to refer to the CDR of an antibody or grafted antibody or variant thereof is intended to be within the scope of the terms defined and used herein. As used herein, the terms "CDR-L1", "CDR-L2" and "CDR-L3" refer to the first CDR, the second CDR and the third CDR in the light chain variable region, respectively. As used herein, the terms "CDR-H1", "CDR-H2" and "CDR-H3" refer to the first CDR, the second CDR and the third CDR in the heavy chain variable region, respectively. As used herein, the terms "CDR-1", "CDR-2" and "CDR-3" refer to the first CDR, the second CDR and the third CDR of the variable region of any chain, respectively.

[0070] Many HVR and CDR delineations have been used, and are covered herein. HVRs for Kabat complementary determining regions (CDRs) are based on sequence variability, and are the most commonly used (Kabat et al., supra). Chothia refers to the position of the structural loops on the contrary (Chothia and Lesk Journal of Molecular Biology 196:901-917 (1987)). AbM HVRs represent a compromise between Kabat CDR and Chothia structural loops, and are used by the AbM antibody modeling software of Oxford Molecular. "Contact" HVRs are based on an analysis of available complex crystal structures. The residues of each HVR from these HVRs are as follows.

[0071]

[0072] HVRs may comprise "extended HVRs" as follows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in VL, and 26-35 (H1), 50-65 or 49-65 (preferred embodiments) (H2), and 93-102, 94-102, or 95-102 (H3) in VH. Variable domain residues are numbered according to Kabat et al. (supra) for each of these extended HVR definitions.

[0073] The Kabat numbering system is generally used when referring to residues in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., "Immune Related Sequences", 5th Edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The "EU numbering system" or "EU index" is generally used when referring to residues in the constant region of an immunoglobulin heavy chain (e.g., Kabat et al., EU index reported in supra). The "EU index in Kabat" refers to the residue numbering of human IgG1 EU antibodies. Unless otherwise specified herein, reference to the number of residues in the variable domain of an antibody means the residue numbering by the Kabat numbering system. Unless otherwise specified herein, reference to the residue number in the constant domain of an antibody means the residue numbering by the EU numbering system (e.g., see U.S. Patent Publication No. 2010-280227).

[0074] As used in this specification, the expression "IGF-1R antibody" should be interpreted as similar to "anti-IGF-1R antibody" and means an antibody capable of binding to IGF-1R. In one embodiment of the present application, the epitope of the antibody is located in the extracellular domain of human IGF-1R (also known as IGF-1R ECD). In a specific embodiment, the antibody or any antigen-binding fragment thereof can be expressed as a protein containing between 10×10 -10 Up to 1×10 -10 and more preferably between 8×10 -10 Up to 2×10 -10 EC between M 50 Binds to IGF-1R.

[0075] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies of the population are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., heteromerization, amidation) that may be present in small amounts. Monoclonal antibodies are highly specific, directed against a single antigenic site. Compared to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

[0076] An "antibody fragment" or "antigen-binding fragment" or "functional fragment" of an antibody comprises a portion of an intact antibody, preferably the antigen-binding region and / or variable region of an intact antibody or an F region of an antibody that retains or has modified FcR binding ability. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; bifunctional antibodies; and linear antibodies (see U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10):1057-1062 (1995)). Additional examples of antibody fragments include antibody derivatives, such as single-chain antibody molecules, monovalent antibodies, and multispecific antibodies formed from antibody fragments.

[0077] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including a native sequence Fc region and a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain may be different, the human IgG heavy chain Fc region is generally defined as extending from the amino acid residue at position Cys226 or Pro230 to its carboxyl terminus. For example, the C-terminal lysine (according to the EU numbering system, residue 447) in the Fc region can be removed during the production or purification of the antibody or by recombinantly engineering the nucleic acid encoding the heavy chain of the antibody. Therefore, the composition of the complete antibody can include an antibody population with all K447 residues removed, an antibody population with no K447 residue removed, and an antibody population with a mixture of antibodies with and without K447 residues. The native sequence Fc region suitable for antibodies of the present disclosure includes human IgG1, IgG2, IgG3, and IgG4.

[0078] A "native sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include native sequence human IgG1 Fc regions (non-A and A allotypes); native sequence human IgG2 Fc regions; native sequence human IgG3 Fc regions; and native sequence human IgG4 Fc regions, as well as naturally occurring variants thereof.

[0079] A "variant Fc region" comprises an amino acid sequence that differs from the amino acid sequence of a native sequence Fc region by at least one amino acid modification (preferably one or more amino acid substitutions). Preferably, the variant Fc region has at least one amino acid substitution compared to the native sequence Fc region or the Fc region of a parent polypeptide, for example, about one to about ten amino acid substitutions and preferably about one to about five amino acid substitutions in the Fc region of a native sequence Fc region or a parent polypeptide. The variant Fc region herein will preferably have at least about 80% homology with the Fc region of a native sequence Fc region and / or a parent polypeptide, and most preferably at least about 90% homology therewith, more preferably at least about 95% homology therewith.

[0080] As used herein, "chimeric antibodies" refer to antibodies (immunoglobulins) in which a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as these antibodies exhibit the desired biological activity (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Nat'l Acad. Sci. USA, 81:6851-55 (1984)). The chimeric antibodies of interest herein include An antibody, wherein the antigen binding region of the antibody is derived from an antibody produced by, for example, immunizing macaques with an antigen of interest. As used herein, "humanized antibodies" are a subset of "chimeric antibodies."

[0081] The "humanized" form of non-human (e.g., mouse) antibodies is a chimeric antibody containing a minimal sequence derived from a non-human immunoglobulin. In certain embodiments, a humanized antibody is a human immunoglobulin (receptor antibody), wherein the residues from the HVR of the recipient are replaced by residues from the HVR of a non-human species (donor antibody) (such as mice, rats, rabbits or non-human primates with desired specificity, affinity and / or ability). In some cases, the FR residues of the human immunoglobulin are replaced by corresponding non-human residues. In addition, a humanized antibody may include residues that are not present in the recipient antibody or the donor antibody. These modifications may be performed to further improve antibody performance, such as binding affinity. In general, a humanized antibody will comprise at least one variable domain and typically substantially all of the variable domains of two variable domains, wherein all or substantially all of the hypervariable loops correspond to loops of non-human immunoglobulin sequences, and all or substantially all of the FR regions are FR regions of human immunoglobulin sequences, but the FR regions may include one or more individual FR residue substitutions that improve antibody performance (such as binding affinity, isomerization, immunogenicity, etc.). The number of these amino acid substitutions in the FR is typically no more than 6 in the H chain and no more than 3 in the L chain. A humanized antibody will optionally also comprise at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin. For additional details, see, e.g., Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, e.g., Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1: 105-115 (1998); Harris, Biochem. Soc. Transactions 23: 1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5: 428-433 (1994); and U.S. Pat. Nos. 6,982,321 and 7,087,409.

[0082] A "human antibody" is an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human and / or an amino acid sequence that has been prepared using any of the techniques disclosed herein for preparing human antibodies. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen binding residues. Human antibodies can be produced using various techniques known in the art, including phage display libraries. Hoogenboom and Winter, Journal of Molecular Biology, 227:381 (1991); Marks et al., Journal of Molecular Biology, 222:581 (1991). Also useful for preparing human monoclonal antibodies are methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147 (1):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001). Human antibodies can be prepared by administering an antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge but whose endogenous loci have been disabled, e.g., an immunized xenogeneic mouse (About XENOMOUSE TM For human antibodies generated by human B cell hybridoma technology, see, e.g., Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006).

[0083] As used herein, an "acceptor human framework" is a framework comprising an amino acid sequence of a VL or VH framework derived from a human immunoglobulin framework or a human consensus framework. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise an amino acid sequence identical thereto, or it may contain pre-existing amino acid sequence changes. In some embodiments, the number of pre-existing amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. When pre-existing amino acid changes are present in VH, preferably, these changes occur only at three, two or one of positions 71H, 73H and 78H; for example, the amino acid residues at these positions may be 71A, 73T and / or 78A. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0084] "Human common framework" is a framework representing the most common amino acid residues in a series of human immunoglobulin VL or VH framework sequences. In general, the human immunoglobulin VL or VH sequences of the series are from a subgroup of variable domain sequences. In general, the subgroup of the sequence is a subgroup such as Kabat et al., "Protein Sequences with Immunological Significance", 5th edition, National Institutes of Health, Bethesda, Maryland, Public Health Service Center (1991). For VL examples include: the subgroup can be subgroup κI, κII, κIII or κIV, such as Kabat et al., supra. In addition, for VH, the subgroup can be subgroup I, subgroup II or subgroup III, such as Kabat et al., supra.

[0085] "Amino acid modification" at a specified position refers to substitution or deletion of a specified residue, or insertion of at least one amino acid residue adjacent to a specified residue. Insertion "adjacent" to a specified residue means insertion within one to two residues thereof. Insertion may be at the N-terminus or C-terminus of a specified residue. A preferred amino acid modification herein is substitution.

[0086] As used herein, "identity" means that at any particular position in the compared sequences, the amino acid residues are identical between the sequences. As used herein, "similarity" means that at any particular position in the compared sequences, the amino acid residues are of similar type between the sequences. For example, leucine can replace isoleucine or valine. Other amino acids that can generally replace each other include, but are not limited to:

[0087] - phenylalanine, tyrosine and tryptophan (amino acids with aromatic side chains);

[0088] - lysine, arginine and histidine (amino acids with basic side chains);

[0089] - Aspartic acid and glutamic acid (amino acids with acidic side chains);

[0090] - Asparagine and glutamine (amino acids with amide side chains); and

[0091] - Cysteine ​​and methionine (amino acids with sulfur-containing side chains).

[0092] The degrees of identity and similarity can be easily calculated. (See, e.g., Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing. Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A.M. and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1993.) York), 1991).

[0093] As used herein, an "interaction" between IGF-1R and a second protein encompasses, but is not limited to, protein-protein interactions, physical interactions, chemical interactions, binding, covalent binding, and ionic binding. As used herein, an antibody "inhibits an interaction between two proteins" when the antibody disrupts, reduces, or completely eliminates the interaction between the two proteins. An antibody or fragment thereof of the present disclosure "inhibits an interaction between two proteins" when the antibody or fragment thereof binds to one of the two proteins.

[0094] As used herein, "amino acid sequence identity" and "homology percentage (%)" with respect to peptide, polypeptide or antibody sequences refer to the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a particular peptide or polypeptide sequence when the sequences are aligned and gaps are introduced, if necessary, to achieve the maximum sequence identity percentage and without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining the percentage of amino acid sequence identity can be achieved in various ways within the capabilities of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGNTM (DNASTAR) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm known in the art required to achieve maximum alignment over the full length of the sequences being compared. The term "prevent" is art-recognized and when used with respect to a condition (e.g., thyroid eye disease (TED)-related symptoms) is relative to a patient not receiving therapy.

[0095] "Recovery" refers to the return to a normal or healthy state. Recovery can be partial (e.g., when the subject returns to a condition less than normal or healthy) or complete (e.g., when the subject returns to a condition that is the same or nearly the same as normal or healthy). An example of a normal or healthy state is a patient's visual acuity prior to thyroid eye disease (TED).

[0096] As used herein, the term "specific recognition" or "specific binding" refers to a measurable and reproducible interaction, such as attraction or binding between a target and an antibody that determines the presence of the target in the presence of a heterogeneous population of molecules (including biomolecules). For example, an antibody that specifically or preferentially binds to a target or epitope is an antibody that binds to this target or epitope with greater affinity, avidity, ease, and / or persistence than it binds to other targets or other epitopes of the target. It should also be understood that, for example, an antibody (or portion) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. Thus, "specific binding" or "preferential binding" does not necessarily require (but it can include) exclusive binding. The association constant of an antibody that specifically binds to a target can be at least about 10 3 M -1 or 10 4 M -1 , sometimes about 10 5 M -1 or 10 6 M -1 In other embodiments, about 10 6 M -1 or 10 7 M -1 , about 10 8 M -1 Up to 10 9 M -1 , or about 10 10 M -1 Up to 10 11 M -1or higher. A variety of immunoassay formats can be used to select antibodies that are specifically immunoreactive with a particular protein. For example, solid phase ELISA immunoassays are routinely used to select monoclonal antibodies that are specifically immunoreactive with a protein. For a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity, see, for example, Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York.

[0097] As used herein, the term "subject" refers to a living mammal and can be used interchangeably with the term "patient". Examples of mammals include, but are not limited to, any member of the class of mammals: humans, non-human primates such as chimpanzees and other ape species and monkey species; farm animals such as cattle, horses, sheep, goats, pigs, etc.; domestic animals such as rabbits, dogs and cats, etc.; laboratory animals, including rodents such as rats, mice and guinea pigs. The term does not denote a particular age or sex.

[0098] The term "therapeutically effective amount" of a compound with respect to the methods of treatment of the present invention refers to an amount of a compound in a formulation that, when administered as part of a desired dosing regimen (to a mammal, preferably a human), alleviates symptoms, improves the condition, or slows the onset of a disease condition, according to clinically acceptable standards for the disorder or condition to be treated or for cosmetic purposes, e.g., at a reasonable benefit / risk ratio applicable to any medical treatment. A therapeutically effective amount herein may vary according to factors such as the disease state, age, sex, and weight of the patient, and the ability of the antibody to elicit a desired response in the individual.

[0099] As used herein, the terms "treat" or "treatment" include reducing, inhibiting or reversing the symptoms, clinical signs or underlying pathology of a condition to stabilize or improve the subject's condition, or to reduce the likelihood that the subject's condition will worsen, as if the subject had not received treatment. "Improving vision" refers to an increase in the ability or state of being able to see, including increased acuity, sensitivity and / or visual field range, relative to pre-treatment.

[0100] Antibody

[0101] In certain aspects, provided herein are pharmaceutical compositions comprising an anti-IGF-1R antibody. Also provided herein are anti-IGF-1R antibodies having a heavy chain comprising a charged amino acid located at the c-terminus thereof.

[0102] The insulin-like growth factor 1 receptor, known as IGF-1R (also known as IGF1R or IGF-IR), is a receptor with tyrosine kinase activity that has 70% homology to the insulin receptor IR. IGF-1R is a glycoprotein with a molecular weight of about 320 kDa. It is a heterotetrameric receptor composed of an extracellular α-subunit and a transmembrane β-subunit, each connected by a disulfide bridge half. IGF-1R binds to IGF1 and IGF2 with a very high affinity (Kd of 1 nM), but is equally capable of binding to insulin with an affinity of 1 / 1000 to 1 / 100 of this affinity. In contrast, IR binds to insulin with a very high affinity, although IGF binds to the insulin receptor with an affinity of only 1 / 100 of this affinity. The tyrosine kinase domains of IGF-1R and IR have very high sequence homology, but the regions with weaker homology involve cysteine-rich regions located on the C-terminal parts of the α-subunit and the β-subunit, respectively. The sequence differences observed in the α-subunits are located in the ligand binding region and are therefore at the origin of the relative affinities of IGF-1R and IR for IGF and insulin, respectively. Differences in the C-terminal part of the β-subunits lead to divergence of the signaling pathways of the two receptors; IGF-1R mediates mitogenic, differentiative and anti-apoptotic effects, whereas activation of the IR involves effects primarily at the level of metabolic pathways.

[0103] Cytoplasmic tyrosine kinase proteins are activated by ligand binding to the extracellular domain of the receptor. The activation of kinases then involves stimulating different intracellular substrates, including IRS-1, IRS-2, Shc and Grb 10. The two main substrates of IGF-1R are IRS and Shc, which mediate most of the growth and differentiation effects associated with IGF attachment to this receptor by activating many effectors downstream. Therefore, the availability of substrates can determine the final biological effects associated with the activation of IGF-1R. When IRS-1 is dominant, cells tend to proliferate and transform. When Shc is dominant, cells tend to differentiate. It seems that the pathway that is mainly involved in the protective effect against apoptosis is the phosphatidylinositol 3-kinase (PI 3-kinase) pathway.

[0104] In certain embodiments, such antibodies exhibit a high ability to internalize upon IGF-1R binding. As used herein, an antibody that is "internalized" or "internalizing" is an antibody that is taken up by a cell (meaning it "enters") upon binding to IGF-1R on a mammalian cell. Certain anti-IGF-1R antibodies provided herein are disclosed in U.S. Pat. No. 10,202,458, which is hereby incorporated by reference for the antibodies, antibody sequences, and related compositions disclosed therein.

[0105] In certain embodiments, the anti-IGF-1R antibody is ronitumumab or an anti-IGF-1R antibody derived from ronitumumab (eg, shares at least one CDR, such as CDRH3, with ronitumumab).

[0106] In certain embodiments, the anti-IGF-1R antibody comprises CDRH1 of SEQ ID NO: 1, CDRH2 of SEQ ID NO: 2, CDRH3 of SEQ ID NO: 3, CDRL1 of SEQ ID NO: 4, CDRL2 of SEQ ID NO: 5, and CDRL3 of SEQ ID NO: 6, as shown in the following table.

[0107] CDR sequence SEQ ID NO CDRH1 GYTFTSYY 1 CDRH2 IWPGDGST 2 CDRH3 ASPMITPNYAMDY 3 CDRL1 QDISKY 4 CDRL2 YTS 5 CDRL3 QQGSTLPYT 6

[0108] In some embodiments, the anti-IGF-1R antibody comprises a heavy chain variable region comprising the following amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWMGWIWPGD GSTKYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYFCASPMITPNYAMDYWGQ GTLVTVSS (SEQ ID NO: 7). In some embodiments, the heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 7. In certain embodiments, the heavy chain variable region comprises a sequence that is identical to SEQ ID NO: 7 except for no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions, additions and / or deletions. In certain embodiments, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, amino acid substitutions, additions and / or deletions occur outside the heavy chain CDR domains.

[0109] In some embodiments, the anti-IGF-1R antibody comprises a light chain variable region comprising the following amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASQDISKYLNWYQQKPGKAPKLLIYYTSRLQSGVP SRFSGRGSGTDYSLTISSLQPEDFATYFCQQGSTLPYTFGGGTKVEIK (SEQ ID NO: 8). In some embodiments, the light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 8. In certain embodiments, the light chain variable region comprises a sequence that is identical to SEQ ID NO: 8 except for no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions, additions and / or deletions. In certain embodiments, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, amino acid substitutions, additions and / or deletions occur outside the light chain CDR domains.

[0110] In some embodiments, an anti-IGF-1R antibody comprises a heavy chain comprising the following amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWMGWIWPGDGSTKYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYFCASPMITPNYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK

[00136] PSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 9). In some embodiments, the heavy chain comprises a sequence as at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9. In certain embodiments, the heavy chain comprises a sequence identical to SEQ ID NO: 9 except for no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions, additions and / or deletions. In certain embodiments, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the amino acid substitutions, additions and / or deletions occur outside the heavy chain CDR domains.

[0111] In some embodiments, the antibody comprises a heavy chain further comprising a charged amino acid located at its c-terminus (e.g., a c-terminal arginine, histidine, lysine, aspartic acid, or glutamic acid). In some embodiments, the charged amino acid is a positively charged amino acid (e.g., arginine, histidine, or lysine). In some embodiments, the charged amino acid is a negatively charged amino acid (e.g., aspartic acid or glutamic acid).

[0112] In some embodiments, an anti-IGF-1R antibody comprises a heavy chain comprising the following amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWMGWIWPGDGSTKYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYFCASPMITPNYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:11).

[0113] In some embodiments, an anti-IGF-1R antibody comprises a heavy chain comprising the following amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWMGWIWPGDGSTKYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYFCASPMITPNYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGH(SEQ ID NO:12).

[0114] In some embodiments, an anti-IGF-1R antibody comprises a heavy chain comprising the following amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWMGWIWPGDGSTKYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYFCASPMITPNYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGR(SEQ ID NO:13).

[0115] In some embodiments, an anti-IGF-1R antibody comprises a heavy chain comprising the following amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWMGWIWPGDGSTKYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYFCASPMITPNYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGD(SEQ ID NO:14).

[0116] In some embodiments, an anti-IGF-1R antibody comprises a heavy chain comprising the following amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWMGWIWPGDGSTKYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYFCASPMITPNYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGE(SEQ ID NO:15).

[0117] In some embodiments, the anti-IGF-1R antibody comprises a light chain comprising the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASQDISKYLNWYQQKPGKAPKLLIYYTSRLQSGVPSRFSGRGSGTDYSLTISSLQPEDFATYFCQQGSTLPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 10). In some embodiments, the light chain comprises a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 10. In certain embodiments, the light chain comprises a sequence identical to SEQ ID NO: 10 except for no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions, additions and / or deletions. In certain embodiments, the amino acid substitutions are conservative amino acid substitutions. In some embodiments, the amino acid substitutions, additions and / or deletions occur outside the heavy chain CDR domains.

[0118] Full-length antibodies can be prepared by using recombinant DNA engineering techniques. Such engineered versions include antibodies produced from natural antibody variable regions, for example, by inserting, deleting or changing the amino acid sequence of a natural antibody or to the amino acid sequence. This type of specific example includes an engineered variable region domain, which contains at least one CDR and optionally one or more framework amino acids from an antibody and the remainder of the variable region domain from a second antibody. The DNA encoding the antibody can be prepared by deleting all of the desired portion of the DNA encoding the full-length antibody. The DNA encoding chimerized antibodies can be prepared by recombining the DNA encoding the variable region of the sequence of the variable region of the variable region of a mammal other than humans substantially or exclusively with the DNA encoding the constant region of the variable region substantially or exclusively derived from the mammal other than humans. The DNA encoding humanized antibodies can be prepared by recombining the DNA encoding the constant region and the variable region except the complementarity determining region (CDR) of the corresponding human antibody region substantially or exclusively derived from the CDR of a mammal other than humans.

[0119] Suitable sources of DNA molecules encoding antibodies include cells expressing full-length antibodies, such as hybridomas. For example, antibodies can be isolated from host cells expressing expression vectors encoding the heavy and / or light chains of antibodies.

[0120] Antibody fragments (including but not limited to Fab fragments) and / or antibody derivatives can also be prepared by using recombinant DNA engineering techniques, which involve manipulation and re-expression of DNA encoding antibody variable and constant regions. Standard molecular biology techniques can be used to modify, add or delete additional amino acids or domains as needed. Any changes to the variable or constant regions are still covered by the terms "variable" and "constant" regions as used herein. In some cases, PCR is used to modify the variable or constant regions by adding or removing the amino acids in the constant region encoding the C H A stop codon was introduced immediately after the codon for the interchain cysteine ​​of 1, making C H The translation of the 1 domain is terminated at the interchain cysteine ​​to generate antibody fragments. Methods for designing suitable PCR primers are well known in the art, and antibody C H The sequence of the 1 domain is also readily available. In some embodiments, a stop codon can be introduced using site-directed mutagenesis.

[0121] The antibodies of the present disclosure can be derived from any antibody isotype ("class"), including, for example, IgG, IgM, IgA, IgD and IgE and subclasses thereof, including, for example, IgG1, IgG2, IgG3 and IgG4. In certain preferred embodiments, the heavy and light chains of the antibodies are from IgG. The heavy and / or light chains of the antibodies can be from mouse IgG or human IgG. In certain other preferred embodiments, the heavy and / or light chains of the antibodies are from human IgG1. In still other preferred embodiments, the heavy and / or light chains of the antibodies are from human IgG4.

[0122] The antibodies disclosed herein can be monoclonal antibodies, polyclonal antibodies, recombinant antibodies, humanized antibodies, human antibodies, chimeric antibodies, multispecific antibodies, antibody fragments thereof, or derivatives thereof. In some embodiments, the antibodies are humanized antibodies.

[0123] The antibody of the present disclosure may also be an antibody fragment, such as a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment, a bifunctional antibody or a single-chain antibody molecule. In some embodiments, the antibody fragment is a Fab fragment.

[0124] In some embodiments, the antibody is a human monoclonal antibody that can be prepared, expressed, generated or isolated by recombinant means, such as (a) an antibody isolated from an animal (e.g., mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom (described further below), (b) an antibody isolated from a host cell transformed to express the antibody (e.g., from a transfectoma), (c) an antibody isolated from a recombinant, combinatorial human antibody library, and (d) an antibody prepared, expressed, generated or isolated by any other means involving splicing of human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline and / or non-germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when using an animal that is transgenic for human Ig sequences, undergo in vivo somatic mutagenesis), and the V of the recombinant antibody is thus H Area and V L The amino acid sequence of the region is derived from human germline V H Sequence and V L Sequences within the human antibody germline repertoire that are related to that sequence but that may not naturally occur in vivo.

[0125] In some embodiments, the antibody is a humanized and / or chimeric monoclonal antibody that can be produced by immunizing rodents (e.g., mice, rats, hamsters, and guinea pigs) with (1) native IGF-1R derived from enzymatic treatment of purified IGF-1R from human plasma or serum or (2) recombinant IGF-1R expressed by a eukaryotic or prokaryotic system or a fragment derived therefrom. Other animals can be used for immunization, such as non-human primates, transgenic mice expressing human immunoglobulins, and severe combined immunodeficiency (SCID) mice transplanted with human B lymphocytes.

[0126] Polyclonal and monoclonal antibodies are naturally produced as immunoglobulin (Ig) molecules in the immune system's response to pathogens. The predominant form in human serum, with a concentration of 8 mg / ml, the approximately 150-kDa IgG1 molecule is composed of two identical heavy chains of approximately 50-kDa and two identical light chains of approximately 25-kDa.

[0127] Hybridomas can be produced by fusing B lymphocytes from immunized animals with myeloma cells by conventional procedures. In addition, antibodies can be produced by screening recombinant single-chain Fv or Fab libraries from human B lymphocytes in a phage display system. The specificity of MAbs for human IGF-1R can be tested by enzyme-linked immunosorbent assay (ELISA), Western immunoblotting or other immunochemical techniques.

[0128] Nucleic acids, vectors and host cells

[0129] Recombinant methods and compositions can be used to produce antibodies suitable for use in the methods of the present disclosure, for example, as described in U.S. Pat. No. 4,816,567. In some embodiments, isolated nucleic acids having nucleotide sequences encoding any of the antibodies of the present disclosure are provided. Such nucleic acids can encode V L / C L The amino acid sequence of the anti-IGF-1R antibody V H / C H 1. In some embodiments, one or more vectors (e.g., expression vectors) containing such nucleic acids are provided. Host cells containing such nucleic acids can also be provided. The host cells can contain the following (e.g., have been transduced with the following): (1) a V vector encoding an antibody; L / C L The amino acid sequence and V containing antibody H / C H 1, or (2) a vector containing a nucleic acid encoding an antibody V L / C L The first vector contains a nucleic acid encoding an amino acid sequence of an antibody V H / C H In some embodiments, the host cell is a eukaryotic cell, for example, a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., a Y0 cell, a NS0 cell, a Sp20 cell). In some embodiments, the host cell is a bacterium, such as Escherichia coli (E. coli).

[0130] Disclosed herein are methods for preparing anti-IGF-1R antibodies. The methods include culturing a host cell of the disclosure containing a nucleic acid encoding an anti-IGF-1R antibody under conditions suitable for expressing the antibody. In some embodiments, the antibody is subsequently recovered from the host cell (or host cell culture medium).

[0131] To recombinantly produce the humanized anti-IGF-1R antibodies of the present disclosure, nucleic acid encoding the antibody is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acid can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that specifically bind to genes encoding the heavy and light chains of the antibody).

[0132] Suitable vectors containing nucleic acid sequences encoding any antibody disclosed herein or polypeptide fragments thereof (including antibodies) described herein include, but are not limited to, cloning vectors and expression vectors. Suitable cloning vectors can be constructed according to standard techniques, or can be selected from a large number of cloning vectors available in the art. Although the selected cloning vector can vary according to the host cell intended to be used, the available cloning vectors generally have the ability to self-replicate, can have a single target for a specific restriction endonuclease, and / or can carry genes that can be used to select markers for clones containing the vector. Suitable examples include plasmids and bacterial viruses (e.g., pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives mpl8, mpl9, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, and shuttle vectors, such as pSA3 and pAT28. These and many other cloning vectors can be obtained from commercial suppliers such as BioRad, Stratagene, and Invitrogen.

[0133] Vectors containing nucleic acids of interest can be introduced into host cells by any of a variety of appropriate means, including electroporation; transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran or other substances; microprojectile bombardment; lipofection; and infection (e.g., in the case where the vector is an infectious agent such as vaccinia virus). The choice of the vector or polynucleotide to introduce will generally depend on the characteristics of the host cell. In some embodiments, the vector contains a nucleic acid containing one or more amino acid sequences encoding an anti-IGF-1R antibody of the present disclosure.

[0134] Suitable host cells for cloning or expressing vectors encoding antibodies include prokaryotes or eukaryotic cells. For example, the anti-IGF-1R antibodies of the present disclosure can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria (e.g., U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523; and Charlton, Methods in Molecular Biology, Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, describes expression of antibody fragments in E. coli). In other embodiments, the antibodies of the present disclosure can be produced in eukaryotic cells (e.g., Chinese hamster ovary (CHO) cells or lymphoid cells (e.g., Y0 cells, NS0 cells, Sp20 cells)) (e.g., U.S. Patent Application No. 14 / 269,950, U.S. Patent No. 8,981,071, Eur J Biochem. Jan. 1, 1991; 195(1):235-42). After expression, the antibody can be isolated from the bacterial cell paste in a soluble fraction, and the antibody can be further purified.

[0135] Pharmaceutical compositions and administration

[0136] The present disclosure generally relates to pharmaceutical compositions comprising the anti-IGF-1R antibodies disclosed herein. As used herein, the expression "pharmaceutical composition" (also referred to as "pharmaceutical formulation") means a combination of at least one active ingredient (e.g., an anti-IGF-1R antibody disclosed herein) and at least one inactive ingredient that is suitable for therapeutic administration to a human or non-human animal when combined with the active ingredient and / or one or more additional inactive ingredients.

[0137] In certain aspects, provided herein is a pharmaceutical composition comprising: (a) at least 75 mg / ml anti-IGF-1R antibody; (b) 20 mM to 30 mM histidine; and (c) 4% to 6% D-sorbitol; wherein the pH of the pharmaceutical composition is 5.5-6.5.

[0138] In certain embodiments, the pharmaceutical composition comprises at least 75 mg / ml of the anti-IGF-1R antibody. In some embodiments, the pharmaceutical composition comprises at least 100 mg / ml, at least 125 mg / ml, at least 150 mg / ml, at least 175 mg / ml, at least 200 mg / ml, or at least 250 mg / ml of the anti-IGF-1R antibody. In certain embodiments, the pharmaceutical composition comprises 75 mg / ml to 300 mg / ml, 100 mg / ml to 300 mg / ml, or 125 mg / ml to 250 mg / ml of the anti-IGF-1R antibody. In some embodiments, the pharmaceutical composition comprises about 125 mg / ml, about 150 mg / ml, about 175 mg / ml, about 200 mg / ml, or about 250 mg / ml of the anti-IGF-1R antibody.

[0139] In some embodiments, the pharmaceutical composition comprises 20mM to 30mM histidine. In certain embodiments, the pharmaceutical composition comprises about 20mM, about 21mM, about 22mM, about 23mM, about 24mM, about 25mM, about 26mM, about 27mM, about 28mM, about 29mM or about 30mM histidine.

[0140] In some embodiments, the pharmaceutical composition comprises 4% to 6% D-sorbitol. In certain embodiments, the pharmaceutical composition comprises about 4%, 5%, or 6% D-sorbitol.

[0141] In some embodiments, the pH of the pharmaceutical composition is 5.5-6.5. In certain embodiments, the pH of the pharmaceutical composition is about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, or about 6.5.

[0142] In some embodiments, the pharmaceutical composition does not contain polysorbate 80. In some embodiments, the pharmaceutical composition contains a small amount of polysorbate 80 (e.g., less than 0.05%). In certain embodiments, the pharmaceutical composition contains no more than 0.05% polysorbate 80. In some embodiments, the pharmaceutical composition contains no more than 0.02% polysorbate 80. In some embodiments, the pharmaceutical composition contains no more than 0.01% polysorbate 80. In some embodiments, the pharmaceutical composition contains no more than 0.005% polysorbate 80. In certain embodiments, the pharmaceutical composition further contains 0.002% to 0.05% polysorbate 80. In some embodiments, the pharmaceutical composition comprises about 0.002%, about 0.003%, about 0.004%, about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.015%, about 0.02%, about 0.025%, about 0.03%, about 0.035%, about 0.04%, about 0.045%, or about 0.05% polysorbate 80.

[0143] In some embodiments, the pharmaceutical composition does not contain poloxamer 188. In some embodiments, the pharmaceutical composition contains a small amount of poloxamer 188 (e.g., less than 0.1%) as a surfactant. In certain embodiments, the pharmaceutical composition contains no more than 0.1% poloxamer 188. In some embodiments, the pharmaceutical composition contains no more than 0.05% poloxamer 188. In some embodiments, the pharmaceutical composition contains no more than 0.02% poloxamer 188. In some embodiments, the pharmaceutical composition contains no more than 0.01% poloxamer 188. In some embodiments, the pharmaceutical composition further contains 0.01% to 0.1% poloxamer 188. In certain embodiments, the pharmaceutical composition contains about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09% or about 0.1% poloxamer 188.

[0144] In certain embodiments, the pharmaceutical composition provided herein shows a high level of stability. As used herein about the pharmaceutical composition, the term "stable" means that after storing a defined time, the antibody in the pharmaceutical composition retains an acceptable degree of structure and / or function and / or biological activity. The composition can be stable, even after storing a defined time, the antibody contained therein does not maintain 100% of its structure and / or function and / or biological activity. In some cases, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98% or about 99% of the structure and / or function and / or biological activity of the antibody maintained after storing a defined time can be considered as "stable".

[0145] Stability can be measured by determining the percentage of natural antibodies retained in the composition after storing a defined time at a given temperature. The percentage of natural antibodies can be determined by size exclusion chromatography (e.g., size exclusion high performance liquid chromatography [SE-HPLC]). As used herein, "acceptable stability" means that at least 90% of the monomeric form of the antibody can be detected in the composition after storing a defined time at a given temperature. In certain embodiments, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the monomeric form of the antibody can be detected in the composition after storing a defined time at a given temperature. The defined time after measuring stable can be at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months or longer. The temperature at which the pharmaceutical composition can be stored when evaluating stability can be any temperature from about -80°C to about 45°C, for example, stored at about -30°C, about -20°C, about 0°C, about 5°C, about 25°C, or about 45°C. For example, if after 3 months of storage at 5°C, greater than about 90%, 95%, 96%, or 97% of the monomeric antibody is detected by SE-HPLC, the pharmaceutical composition can be considered stable. If after 6 months of storage at 5°C, greater than about 90%, 95%, 96%, or 97% of the monomeric antibody is detected by SE-HPLC, the pharmaceutical composition can be considered stable. If after 9 months of storage at 5°C, greater than about 90%, 95%, 96%, or 97% of the monomeric antibody is detected by SE-HPLC, the pharmaceutical composition can be considered stable. If after 3 months of storage at 25°C, greater than about 90%, 95%, 96%, or 97% of the monomeric antibody is detected by SE-HPLC, the pharmaceutical composition can also be considered stable. The pharmaceutical composition may also be considered stable if greater than about 90%, 95%, 96%, or 97% of the monomeric antibody is detected by SE-HPLC after 6 months storage at 25° C. The pharmaceutical composition may also be considered stable if greater than about 90%, 95%, 96%, or 97% of the monomeric antibody is detected by SE-HPLC after 9 months storage at 25° C.

[0146] Other methods can be used to assess the stability of the pharmaceutical compositions disclosed herein, such as differential scanning calorimetry (DSC) for determining thermal stability, controlled stirring for determining mechanical stability, and absorbance at about 350 nm or about 405 nm for determining solution turbidity. For example, if the OD of a composition is less than 0.01 after storage at about 5° C. to about 25° C. for 6 or more months, the composition may be evaluated for stability. 405 Relative to the OD of the composition at t=0 405 The pharmaceutical compositions disclosed herein can be considered stable if the change in % is less than about 0.05 (e.g., 0.04, 0.03, 0.02, 0.01, or less).

[0147] Stability can also be assessed by measuring the biological activity and / or binding affinity of the antibody to its target. For example, if after storage for a defined amount of time (e.g., 1 month to 12 months) at, e.g., 5°C, 25°C, 45°C, etc., the anti-IGF-1R antibody contained within the composition binds to IGF-1R with an affinity of at least 50%, 60%, 70%, 80%, 90%, 95% or greater relative to the binding affinity of the antibody prior to said storage, then the compositions disclosed herein can be considered stable. Additional methods for assessing the stability of an antibody in a composition are shown in the examples presented below.

[0148] In certain embodiments, after 12 weeks at -20°C and 2-8°C, the pharmaceutical compositions provided herein continue to show very good stability in terms of low aggregation; confirmed by both visual assessment and SEC-HPLC. Data from SEC-HPLC revealed that there was no significant increase in high molecular weight species (HMWS) at T0-T12 weeks, with two temperatures performing similarly. CEX-HPLC analysis revealed that after 12 weeks at -20°C and 2-8°C, the composition showed good chemical stability. From the CEX-HPLC data, slightly higher values ​​of the main species% were observed for samples kept at 2-8°C. This indicates that the freeze-thaw cycles experienced by the sample at -20°C do not affect aggregation, but are detrimental to the chemical stability of the molecule. Improvements in chemical stability were obtained from formulation 3a (seen at both 2-8°C and -20°C in Example 1), which contained sorbitol as a tension regulator without the addition of PS80.

[0149] DLS measurements revealed that after 12 weeks at both temperatures, the particle size Z D PDI values ​​showed an increasing trend over time, but all compositions remained monodisperse after 12 weeks.

[0150] In some embodiments, the pharmaceutical composition is stable for at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, at least 13 weeks, at least 14 weeks, at least 15 weeks, or at least 16 weeks. In some embodiments, the pharmaceutical composition is stable at a temperature of -20°C to 8°C.

[0151] In fluid form, the pharmaceutical composition provided herein can show low to medium levels of viscosity in certain embodiments. As used herein, "viscosity" can be "kinematic viscosity" or "absolute viscosity". "Kinematic viscosity" is a measure of the resistance flow of a fluid under the influence of gravity. When two equal volumes of fluid are placed in the same capillary viscometer and allowed to flow by gravity, the time that a viscous fluid takes to flow through a capillary is longer than the time that a less viscous fluid takes to flow through a capillary. For example, if a fluid takes 200 seconds to complete its flow, and another fluid takes 400 seconds, then on the kinematic viscosity scale, the viscosity of the second fluid is twice the viscosity of the first fluid. "Absolute viscosity", sometimes referred to as dynamic viscosity or simple viscosity, is the product of kinematic viscosity and fluid density (absolute viscosity=kinematic viscosity × density). The dimension of kinematic viscosity is L 2 / T, where L is the length and T is the time. Typically, kinematic viscosity is expressed in centistokes (cSt). The SI unit for kinematic viscosity is square millimeters per second, or 1 cSt. Absolute viscosity is expressed in centipoise (cP). The SI unit for absolute viscosity is milliPascal seconds (mPa·s), where 1 cP = 1 mPa·s.

[0152] As used herein, a low viscosity level for a pharmaceutical composition disclosed herein will exhibit an absolute viscosity of less than about 20 centipoise (cP). For example, if the composition exhibits an absolute viscosity of about 19 cP, about 18 cP, about 17 cP, about 16 cP, about 15 cP, about 14 cP, about 13 cP, about 12 cP, about 11 cP, about 10 cP, about 9 cP, about 8 cP, about 7 cP, about 6 cP, about 5 cP, about 4 cP or less at 21 ° C, the pharmaceutical composition disclosed herein will be considered to have "low viscosity". As used herein, a medium viscosity level for a pharmaceutical composition disclosed herein will exhibit an absolute viscosity between about 30 cP and about 20 cP at 21 ° C. For example, a pharmaceutical composition disclosed herein would be considered to have "medium viscosity" if the composition exhibits an absolute viscosity of about 30 cP, about 29 cP, about 28 cP, about 27 cP, about 26 cP, about 25 cP, about 24 cP, about 23 cP, about 22 cP, about 21 cP, or about 20 cP at 21 °C when measured using standard viscosity measurement techniques.

[0153] In some embodiments, the osmotic pressure of the pharmaceutical composition is within the physiological osmotic pressure range of 250-400 mOsm / kg. In some embodiments, the viscosity of the pharmaceutical composition does not exceed 30 cP at 21°C. In some embodiments, the viscosity of the pharmaceutical composition does not exceed 15 cP at 21°C. In some embodiments, the viscosity of the pharmaceutical composition is about 10 cP, about 11 cP, about 12 cP, about 13 cP, about 14 cP, about 15 cP, about 16 cP, about 17 cP, about 18 cP, about 19 cP, about 20 cP, about 21 cP, about 22 cP, about 23 cP, about 24 cP, about 25 cP, about 26 cP, about 27 cP, about 28 cP, about 29 cP or about 30 cP at 21°C.

[0154] syringe

[0155] In certain aspects, the disclosure relates to a syringe comprising a pharmaceutical composition disclosed herein. In some embodiments, the syringe comprises a delivery volume of no more than 2 ml. In some embodiments, the syringe comprises a needle of no more than 24G (e.g., 25G, 27G). In some embodiments, the syringe is an automated reusable fixed dose pen. In some embodiments, the syringe is an automated reusable variable dose pen. In some embodiments, the syringe is an automated disposable fixed dose auto-injector.

[0156] In certain embodiments, the pharmaceutical composition provided herein can be contained in any container suitable for storing medicine and other therapeutic compositions. For example, the pharmaceutical composition can be contained in a sealed and sterilized plastic or glass container with a defined volume, such as a vial, an ampoule, a syringe, a barrel or a bottle. Different types of vials can be used to hold the composition provided herein, and these vials include, for example, clear and opaque (e.g., amber) glass or plastic vials. Similarly, any type of syringe can be used to hold and / or apply pharmaceutical compositions disclosed herein.

[0157] The pharmaceutical composition provided herein can be contained in a "normal tungsten" syringe or a "low tungsten" syringe. As will be appreciated by those of ordinary skill in the art, the process of making a glass syringe generally involves the use of a hot tungsten rod, which acts to pierce the glass, thereby creating a hole through which liquid can be drawn and discharged from the syringe. This process results in trace amounts of tungsten being deposited on the inner surface of the syringe. Subsequent washing and other processing steps can be used to reduce the amount of tungsten in the syringe. As used herein, the term "normal tungsten" means that the syringe contains tungsten greater than 500 parts per billion (500 ppb). The term "low tungsten" means that the syringe contains less than 500 ppb of tungsten. For example, a low tungsten syringe can contain less than about 490 ppb, 480 ppb, 470 ppb, 460 ppb, 450 ppb, 440 ppb, 430 ppb, 420 ppb, 410 ppb, 390 ppb, 350 ppb, 300 ppb, 250 ppb, 200 ppb, 150 ppb, 100 ppb, 90 ppb, 80 ppb, 70 ppb, 60 ppb, 50 ppb, 40 ppb, 30 ppb, 20 ppb, 10 ppb, or less ppb of tungsten.

[0158] The pharmaceutical composition provided herein can be contained in a plastic syringe. In the past decade, pharmaceutical proteins and peptide drug products have been approved for use with prefilled plastic syringes. For example, the pharmaceutical composition can be contained in a Crystal Zenith (CZ) syringe (Daikyo Seiko, Tokyo).

[0159] The rubber plunger used in the syringe and the rubber stopper used to close the opening of the vial can be coated to prevent the drug contents of the syringe or vial from being contaminated and / or to maintain its stability. Therefore, according to certain embodiments, the pharmaceutical composition provided herein can be contained in a syringe comprising a coated plunger or in a vial sealed with a coated rubber stopper. For example, the plunger or stopper can be coated with a fluorocarbon film. Examples of coated stoppers and / or plungers suitable for use with vials and syringes containing the pharmaceutical compositions disclosed herein are mentioned in, for example, U.S. Patents Nos. 4,997,423; 5,908,686; 6,286,699; 6,645,635; and 7,226,554, the contents of which are hereby incorporated by reference in their entirety. Specific exemplary coated rubber stoppers and plungers that can be used in the methods disclosed herein can be found under the trade name Commercially available from West Pharmaceutical Services, Inc. (Lionville, Pa.).

[0160] In certain embodiments, the pharmaceutical composition can be administered to a patient by a parenteral route such as injection (e.g., subcutaneous, intravenous, intramuscular, intraperitoneal, suborbital, intravitreal, intraocular, subconjunctival, retrobulbar, peribulbar, and / or intrathecal injection). A number of reusable pens and / or automatic syringe delivery devices can be used to deliver the pharmaceutical compositions disclosed herein subcutaneously. Examples include, but are not limited to, AUTOPEN TM (Owen Mumford, Inc., Woodstock, UK), DISETRONIC TM Pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25 TM Pen, HUMALOG TM Pen, HUMALIN 70 / 30 TM PEN (Eli Lilly and Co., Indianapolis, Ind.), NOVOPEN TM I, II and III (NovoNordisk, Copenhagen, Denmark), NOVOPEN JUNIOR TM (Novo Nordisk, Copenhagen, Denmark), BD TM Pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN TM 、OPTIPEN PRO TM 、OPTIPEN STARLET TM and OPTICLIK TM (sanofi-aventis, Frankfurt, Germany), to name a few. Examples of disposable pen and / or auto-injector delivery devices for subcutaneous delivery of pharmaceutical compositions disclosed herein include, but are not limited to, SOLOSTAR TM Pen (Sanofi-Aventis), FLEXPEN TM (Novo Nordisk) and KWIKPEN TM (Eli Lilly), SURECLICK TMAutoinjector (Amgen, Thousand Oaks, Calif.), PENLET TM (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA TM Pen (Abbott Labs, Abbott Park, Ill.), to name a few.

[0161] It is also contemplated herein that the pharmaceutical compositions disclosed herein are delivered using West's SelfDose and SmartDose injection systems. It is also contemplated herein that the pharmaceutical compositions disclosed herein are delivered using microinfusion devices. As used herein, the term "microinfusion device" means a subcutaneous delivery device designed to slowly administer a large volume (e.g., up to about 2.5 mL or more) of the therapeutic composition over an extended period of time (e.g., about 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes or more minutes). See, for example, U.S. Patent No. 6,629,949; No. 6,659,982; and Meehan et al., J. Controlled Release 46: 107-116 (1996).

[0162] Treatment

[0163] In certain aspects, the present disclosure relates to methods of preventing, reducing the risk of developing, or treating diseases or conditions associated with IGF-1R, comprising administering a pharmaceutical composition and / or an anti-IGF-1R antibody disclosed herein. The present disclosure also generally relates to methods of preventing, reducing the risk of developing, or treating diseases and conditions associated with IGF-1R, comprising administering an anti-IGF-1R antibody disclosed herein. The present disclosure also generally relates to methods of preventing, reducing the risk of developing, or treating diseases and conditions associated with IGF-1R, comprising administering a pharmaceutical composition using a syringe disclosed herein. In some embodiments, the disease or condition is thyroid eye disease (TED).

[0164] In some embodiments, the disease or disorder is stroke, acromegaly, diabetic nephropathy (diabetic kidney disease), idiopathic pulmonary fibrosis, interstitial lung disease, obesity, type 2 diabetes, juvenile idiopathic arthritis (JIA), diffuse cutaneous systemic sclerosis, Sjögren's syndrome calcification and vasculitis, cachexia and sarcopenia, diabetic macular edema, atherosclerosis, peripheral arterial disease (PAD), myocardial infarction and stroke, diabetic foot and skin, rheumatoid arthritis, neurofibromatosis 1, neurofibromatosis 2, polycystic kidney disease, polycystic liver disease, polycystic ovary syndrome, Alzheimer's disease, cognitive decline, dementia, depression and anxiety, asthma, aging, thyroid eye disease, idiopathic orbital inflammation, human type 2 lipodystrophy and related cardiomyopathy, autosomal dominant polycystic kidney disease (ADPKD), NASH, Graves' disease and / or Hashimoto's thyroiditis.

[0165] In some embodiments, the disease or disorder is thyroid eye disease (TED). Thyroid associated eye disease (TAO), also known as thyroid eye disease (TED), Graves' ophthalmopathy or orbitopathy (GO), thyrotoxic exophthalmos, thyroid dysfunction eye disease and several other terms, is an orbital disease associated with thyroid dysfunction. TED is divided into two types. Active TED, which typically lasts for 1-3 years, is characterized by a persistent autoimmune / inflammatory response in the soft tissue of the orbit. Active TED causes expansion and remodeling of the soft tissue of the eye. The autoimmune / inflammatory response of active TED will spontaneously subside, and the condition will turn into inactive TED. Inactive TAO is a term used to describe the long-term / permanent sequelae of active TED. The cause of TED is unknown. TED is usually associated with Graves' hyperthyroidism, but can also occur as part of other autoimmune conditions that affect the thyroid gland and produce pathology in the orbital and periorbital tissues, and rarely in the pretibial skin (pretibial myxedema) or toes (thyroid clubbing). TED is an autoimmune orbital disease in which the orbit and periocular soft tissues are primarily affected, with secondary effects on the eye and vision. In TED, the eye is forced forward (protrudes) out of the socket due to inflammation and expansion of the orbital soft tissues (primarily ocular muscles and fat) - a phenomenon known as proptosis or exophthalmos. Although most cases of TED do not result in vision loss, the condition can lead to sight-threatening exposure keratopathy, troublesome diplopia (double vision), and compressive thyroid dysfunction optic neuropathy. TED may occur before, at the same time, or after systemic complications of thyroid dysfunction. The ocular manifestations of TED include upper eyelid retraction, eyelid lag, swelling, redness (erythema), conjunctivitis and proptosis (exophthalmos or proptosis), conjunctival chemosis, periorbital edema, and changes in eye movements, as well as significant functional, social, and cosmetic consequences. Many of the signs and symptoms of TED, including proptosis and ocular hyperemia, result from the expansion of orbital adipose tissue and periocular muscles. Adipose tissue volume increases in part due to the development of new adipocytes (adipogenesis) within the orbital fat. Accumulation of hydrophilic glycosaminoglycans (primarily hyaluronic acid) within the perimembranous connective tissue between the orbital adipose tissue and extraocular muscle fibers further expands the fat compartment and enlarges the extraocular muscle body. Hyaluronic acid is produced by fibroblasts resident within the orbital fat and extraocular muscles, and its synthesis in vitro is stimulated by several cytokines and growth factors, including IL-1β, interferon-γ, platelet-derived growth factor, thyroid-stimulating hormone (TSH), and insulin-like growth factor I (IGF-I).

[0166] Antibodies that activate the insulin-like growth factor I receptor (IGF-IR) have also been detected and implicated in active TED. Without being bound by any theory, it is believed that the TSHR and IGF-IR form a physical and functional complex in orbital fibroblasts, and blocking the IGF-IR appears to attenuate both IGF-1 and TSH dependent signaling. It has been suggested that blocking IGF-IR using antibody antagonists may reduce both TSHR and IGF-I dependent signaling and thus interrupt the pathological activity of autoantibodies that act as agonists at either receptor.

[0167] IGF-IR is a ubiquitously expressed heterotetrameric protein that is involved in regulating proliferation and metabolic function in many cell types. It is a tyrosine kinase receptor containing two subunits. IGF-IRα contains a ligand binding domain, while IGF-IRβ is involved in signal transduction and contains tyrosine phosphorylation sites.

[0168] Thyroid eye disease (TED) is a debilitating autoimmune disorder that occurs in patients with Graves' disease in which inflammation in the muscle and fatty tissue behind the eye leads to proptosis, diplopia, redness, pain, and swelling, resulting in light sensitivity, blurred vision, and in severe cases, blindness. The mechanistic basis of TED involves a complex interaction between autoantibody-mediated stimulation of thyroid stimulating hormone receptor (TSHR) and insulin-like growth factor 1 receptor (IGF-1R) signaling in orbital fibroblasts, which results in inflammation and dilation of orbital tissue. Current therapies include corticosteroids and teprotumumab as well as surgical interventions to prevent vision loss. Teprotumumab is a high-affinity (K-1) inhibitor directed against IGF-1R. D <50 pM) monoclonal antibody that induces rapid and efficient receptor internalization. Ronituzumab is being developed as a potential treatment for TED. To support the clinical development of ronituzumab, a multicolor flow cytometric assay was developed to monitor the binding of ronituzumab to IGF-1R on the surface of human peripheral blood mononuclear cells (PBMCs).

[0169] Ronituzumab is a humanized monoclonal antibody against IGF-1R. Ronituzumab has high affinity and specificity for IGF-1R. Specifically, Ronituzumab has picomolar affinity for IGF-1R. In certain preferred embodiments, the k of the binding epitope of Ronituzumab to IGF-1R is DThe amount of ronituzumab in the serum is about 30 pM. When ronituzumab binds to IGF-1R, it induces receptor internalization, which results in signal blocking with IGF-1R, which can cause the therapeutic effect of ronituzumab. The IGF-1R internalization can occur within a few minutes after the administration of the pharmaceutical composition comprising ronituzumab. In certain preferred embodiments, the therapeutically effective serum concentration of ronituzumab can be obtained within about one hour after the administration of the pharmaceutical composition comprising ronituzumab.

[0170] Ronituzumab has unique and beneficial pharmacological properties. In certain preferred embodiments, the administration of a pharmaceutical composition comprising Ronituzumab induces greater than about 70% IGF-1R internalization. In certain preferred embodiments, the administration of a pharmaceutical composition comprising Ronituzumab induces greater than about 80% IGF-1R internalization. In certain preferred embodiments, the administration of a pharmaceutical composition comprising Ronituzumab induces greater than about 85% IGF-1R internalization. In certain preferred embodiments, the administration of a pharmaceutical composition comprising Ronituzumab induces greater than about 90% IGF-1R internalization. In certain preferred embodiments, the administration of a pharmaceutical composition comprising Ronituzumab induces greater than about 95% IGF-1R internalization. In certain preferred embodiments, the administration of a pharmaceutical composition comprising Ronituzumab induces greater than about 97% IGF-1R internalization. In certain preferred embodiments, the administration of a pharmaceutical composition comprising Ronituzumab induces greater than about 99% IGF-1R internalization.

[0171] In certain embodiments of the compositions and methods provided herein, the pharmaceutical composition is formulated for subcutaneous administration. In certain embodiments, the pharmaceutical composition is administered subcutaneously. In certain embodiments, the pharmaceutical composition is formulated for intramuscular administration. In certain embodiments, the pharmaceutical composition is administered intramuscularly. In certain embodiments, the pharmaceutical composition is formulated for suborbital, intravitreal, intraocular, subconjunctival, retrobulbar, periorbital and / or intrathecal administration. In certain embodiments, the pharmaceutical composition is administered by suborbital, intravitreal, intraocular, subconjunctival, retrobulbar, periorbital and / or intrathecal injection.

[0172] In certain embodiments, the pharmaceutical composition is administered in a delivery volume of no more than 3 ml, 2.5 ml, 2 ml, 1.5 ml, or 1 ml. In some embodiments, the pharmaceutical composition is administered in a delivery volume of no more than 2 ml.

[0173] In certain embodiments, the pharmaceutical composition is administered through a needle no larger than 24G, 25G, or 27G in size. In some embodiments, the pharmaceutical composition is administered in a 24G needle, a 25G needle, or a 27G needle.

[0174] In certain embodiments, the pharmaceutical composition is administered with an injection force of no more than 14 N, 13 N, 12 N, 11 N, 10 N, 9 N, 8 N, 7 N, or 6 N. In certain embodiments, the pharmaceutical composition is administered with an injection force of no more than 12 N. In certain embodiments, the pharmaceutical composition is administered with an injection force of about 4 N, about 5 N, about 6 N, about 7 N, about 8 N, about 9 N, about 10 N, about 11 N, or about 12 N.

[0175] In some embodiments, the method reduces the severity of the thyroid eye disease (TED). In some embodiments, the method reduces proptosis in an eye of a subject with thyroid eye disease (TED). In some embodiments, the proptosis is reduced by at least 2 mm. In some embodiments, the proptosis is reduced by at least 3 mm. In some embodiments, the proptosis is reduced by at least 4 mm.

[0176] In some embodiments, the method reduces the clinical activity score (CAS) of thyroid eye disease (TED). In some embodiments, the clinical activity score (CAS) is reduced by at least 2 points. In some embodiments, the clinical activity score (CAS) is reduced to one (1). In some embodiments, the clinical activity score (CAS) of the subject is reduced to zero (0). In some embodiments, the method improves the quality of life of the subject. In some embodiments, the quality of life is measured by the Graves' Ophthalmopathy Quality of Life (GO-QoL) assessment. In some embodiments, the quality of life is measured by its visual function or appearance subscale. In some embodiments, the quality of life is measured by the European Graves' Orbitopathy Group (EUGOGO) guidelines. In some embodiments, the method reduces the severity of diplopia. In some embodiments, the diplopia is persistent diplopia. In some embodiments, the diplopia is non-persistent diplopia. In some embodiments, the diplopia is intermittent diplopia.

[0177] In certain aspects, the present invention provides methods of administering pharmaceutical compositions comprising ronituzumab. These pharmaceutical compositions can be administered to patients suffering from thyroid eye disease (TED). In certain embodiments, the pharmaceutical compositions of the present invention can be administered intravenously. The methods of the present invention provide for intravenous infusion of a pharmaceutical composition comprising up to about 3.0 mg / kg ronituzumab to the patient. In certain embodiments, the methods of the present invention provide for administering a pharmaceutical composition comprising about 0.1 mg / kg, 0.3 mg / kg, 1.0 mg / kg or 3.0 mg / kg ronituzumab in the form of an intravenous infusion. The present invention further provides that the intravenous infusion can occur for a period of about 15 minutes to about 120 minutes. In certain preferred embodiments, the intravenous infusion occurs for a duration of about 60 minutes.

[0178] In certain aspects, the present invention provides a pharmaceutical composition comprising ronitumumab administered in the form of a subcutaneous injection. In certain embodiments, the method of the present invention provides subcutaneous administration of up to about 250 mg ronitumumab. In certain embodiments, the pharmaceutical composition for subcutaneous administration comprises about 20 mg, 40 mg, 125 mg or 250 mg ronitumumab. The volume of the pharmaceutical composition for subcutaneous administration can be about 0.5 mL to about 3 mL. In a preferred embodiment, the volume administered is up to about 2 mL. Beneficially, since subcutaneous administration is performed within a tolerable volume, the pharmaceutical composition can be administered in an outpatient setting, or the pharmaceutical composition of the present invention can be administered by the patient himself. This creates a convenient and efficient way for administering a pharmaceutical composition comprising ronitumumab. Specifically, compared to intravenous administration, subcutaneous administration of a pharmaceutical composition is beneficial due to the ease of administering the pharmaceutical composition.

[0179] In certain embodiments of the invention, administration of a pharmaceutical composition comprising ronitumumab maximizes the IGF-1R receptor occupancy of an anti-IGF-1R antibody. This maximum IGF-1R occupancy can be achieved at any time after administration of a pharmaceutical composition comprising ronitumumab. In certain embodiments, this is achieved after about 12 hours after administration of a pharmaceutical composition comprising ronitumumab. The administered pharmaceutical composition may comprise from about 100 mg to about 300 mg of ronitumumab. In a preferred embodiment, the pharmaceutical composition may comprise about 125 mg or about 250 mg of ronitumumab. It is noteworthy that the maximum IGF-1R receptor occupancy can be maintained for up to several months after a single administration. In a preferred embodiment, the IGF-1R receptor occupancy is maintained for at least about 4 weeks after a single administration of a pharmaceutical composition comprising ronitumumab. The maximum receptor occupancy level can be maintained after a single subcutaneous injection or intravenous infusion of a pharmaceutical composition comprising ronitumumab.

[0180] Titumumab (i.e., a previously known antibody for IGF-1R occupancy) is maintained at a concentration of 200 μg / mL to improve efficacy. The efficacy of ronitumab is about 75 times that of tetumumab, and the equivalent serum concentration required to maintain the therapeutic activity of ronitumab is predicted to be about 3 μg / mL. In certain aspects, the serum concentration required for maximum IGF-1R receptor internalization is a concentration of approximately about 3 μg / mL ronitumab. Therefore, the therapeutically effective serum concentration of ronitumab is at least about 1 μg / mL. In certain preferred embodiments, the therapeutically effective serum concentration of ronitumab is at least about 3 μg / mL. Importantly, the pharmaceutical composition of the present invention provides a therapeutically effective serum concentration of ronitumab to the patient.

[0181] In certain aspects of the invention, the pharmaceutical composition comprising ronitumumab administered to the patient is safe and tolerable. In addition, the pharmaceutical composition of the invention has minimal adverse events.

[0182] In certain aspects, the present invention provides methods of treating thyroid eye disease (TED), comprising subcutaneously administering a pharmaceutical composition comprising about 10 mg to about 500 mg of ronituzumab. In certain embodiments, the pharmaceutical composition comprises about 20 mg of ronituzumab. In certain embodiments, the pharmaceutical composition comprises about 40 mg of ronituzumab. In certain embodiments, the pharmaceutical composition comprises about 125 mg of ronituzumab. In certain embodiments, the pharmaceutical composition comprises about 250 mg of ronituzumab.

[0183] In certain embodiments, the pharmaceutical composition comprising ronituzumab is administered once a week. In certain embodiments, the pharmaceutical composition comprising ronituzumab is administered twice a week. In certain embodiments, the pharmaceutical composition comprising ronituzumab is administered three times a week. In certain embodiments, the pharmaceutical composition comprising ronituzumab is administered every other day (i.e., once every two days). In certain preferred embodiments, the pharmaceutical composition comprising ronituzumab is administered once every two (2) weeks. In certain preferred embodiments, the pharmaceutical composition comprising ronituzumab is administered once every three (3) weeks. In certain preferred embodiments, the pharmaceutical composition comprising ronituzumab is administered once every four (4) weeks. In certain embodiments, the pharmaceutical composition comprising ronituzumab is administered once every five (5) weeks. In certain embodiments, the pharmaceutical composition comprising ronituzumab is administered once every six (6) weeks.

[0184] In certain preferred embodiments, the pharmaceutical composition comprising ronituzumab is administered on day 1 and day 14. In other preferred embodiments, the pharmaceutical composition comprising ronituzumab is administered on day 1 and day 21. In other preferred embodiments, the pharmaceutical composition comprising ronituzumab is administered on day 1 and day 28.

[0185] Examples

[0186] Example 1: Development of anti-human IGF-1R mAb before formulation

[0187] A drug product formulation of a high dose of 125 mg / mL anti-IGF-1R antibody was prepared in which the concentration of active pharmaceutical ingredient (API) was higher than the concentration of drug substance at 20 mg / mL. The composition of excipients remained the same in both the 20 mg / mL formulation and the 125 mg / mL formulation.

[0188] The anti-IGF-1R antibody is formulated in an isotonic sterile solution manufactured at two concentrations, 20 mg / mL and 125 mg / mL, to enable delivery by intravenous and subcutaneous administration. The composition of the drug product is identical to the composition of the drug substance in both product formulations. The drug product is presented in single-dose depyrogenated Type 1 glass vials.

[0189] Process 1 drug substance was prepared at 20 mg / mL in formulation buffer containing 25 mM histidine, 6% sucrose at pH 6.0. There was no process development performed for the manufacture of the 20 mg / mL batch. The process consisted of sterile filtration through two filters installed in series and a fill-finish operation.

[0190] For 125 mg / mL drug product (DP) production, manufacturing process development was performed to optimize the key parameters of the ultrafiltration process used to concentrate the antibody to a 125 mg / mL concentration, followed by sterile filtration through two in-line filters and fill-finish operations. During process development, technical batches of 125 mg / mL product concentration were manufactured.

[0191] L-histidine is used in a concentration of 25 mM in pharmaceutical formulations as a pH buffer. Sucrose is a stabilizer and also an osmotic pressure regulator (tonicity agent). It can protect the active ingredient from degradation and maintain the osmotic pressure of the pharmaceutical product. Hydrochloric acid is used as a pH regulator. Water is used as a solvent.

[0192] The data obtained to date indicate that the above excipients in this formulation are highly effective in maintaining the stability of the drug product and do not adversely affect the quality attributes of the drug product.

[0193] Development of formulations

[0194] In the early formulation development stage, several buffer formulations (25 mM glutamate, 25 mM acetic acid, 25 mM histidine and 25 mM phosphate) were tested across a range of pH (4.5-7.5) and in combination with NaCl (150 mM), sucrose (9%) and polysorbate 80 (0.02%). Fourteen (14) formulations were prepared and further investigated. The following tests were performed during the pre-formulation development stage: visual appearance, protein content, CE-SES (reduced and non-reduced), SEC-HPLC, CEX-HPLC and DLS.

[0195] Initial screening studies were performed to select formulation conditions for the humanized anti-IGF-1R antibody that would allow good stability based on defined analytical criteria.

[0196] The purpose of the screening was to make a limited selection of four (4) formulations. In the initial screening study, the effects of the following four factors were investigated: (i) the nature of the buffer; 25 mM histidine or 25 mM phosphate; (ii) pH: pH 6-pH7; (iii) NaCl concentration: 0-150 mM; and (iv) Polysorbate 80 concentration: 0-0.1% v / v.

[0197] Two experimental plans (one for each buffer) were set up to enable modeling of the response surface for each plan.Central points were analyzed in triplicate to estimate experimental variability.

[0198] Two different experimental plans (three complex matrices with triplicate center points) were set up in two different buffers as follows:

[0199] 25 mM Histidine Buffer Experimental Plan (See Table 1)

[0200] 25 mM phosphate buffer experimental plan (see Table 2)

[0201] Table 1: Experimental plan for 25 mM histidine buffer

[0202]

[0203]

[0204] Table 2: Experimental plan for 25 mM phosphate buffer

[0205] Preparation No. Buffer pH NaCl(mM) Polysorbate 80 (% v / v) P1 25 mM phosphate 6.0 0 0 P2 25 mM phosphate 7.0 0 0 P3 25 mM phosphate 6.0 150 0 P4 25 mM phosphate 7.0 150 0 P5 25 mM phosphate 6.0 0 0.10 P6 25 mM phosphate 7.0 0 0.10 P7 25 mM phosphate 6.0 150 0.10 P8 25 mM phosphate 7.0 150 0.10 P9 25 mM phosphate 6.0 75 0.05 P10 25 mM phosphate 7.0 75 0.05 P11 25 mM phosphate 6.5 0 0.05 P12 25 mM phosphate 6.5 150 0.05 P13 25 mM phosphate 6.5 75 0 P14 25 mM phosphate 6.5 75 0.10 P15 25 mM phosphate 6.5 75 0.05 P16 25 mM phosphate 6.5 75 0.05 P17 25 mM phosphate 6.5 75 0.05

[0206] The experimental plan implemented was run using specialized software applications (Nemrod and MODDE) that perform statistical analysis of the data to verify the validity and relevance of the generated model. These tools make it possible to see the influence of the factors tested on the studied response by plotting the response surface and calculating the optimal conditions according to defined criteria.

[0207] The study responses for each plan are: (i) the melting temperature T at T0 in °C m1 and T m2 (ii) the increase in the rate of acidic variants after stress at 40°C for 10 days (CIEX analysis); and (iii) the rate of multimers after stress at 40°C for 10 days (SEC analysis).

[0208] Analysis of the histidine program showed that pH had a significant effect on all three responses: high pH (pH 7) resulted in a significant decrease in T m , polymer rate and acidic variant rate increased. The presence of NaCl increased T mand polymer rates, but tended not to result in excessive acidic variants. Finally, polysorbate 80 had little effect on the three responses; at the percentages tested, its presence would even tend to favor the appearance of polymers. The response surface is plotted as follows ( Figure 1-4 ).

[0209] To minimize the multimer rate, it is preferred to use pH 6-6.5 and <100 mM salt. At the percentages tested, the addition of polysorbate 80 was undesirable because the presence of the surfactant led to an increase in the multimer rate.

[0210] In order to limit the appearance of acidic variants, it is necessary again to adopt a rather low pH (6-6.5); the presence of NaCl helps to stabilize this standard. However, the presence of polysorbate 80 has no significant effect.

[0211] In order to facilitate high T m values, pH 7 is preferred, and polysorbate 80 is not desirable at the percentages tested.

[0212] For 15 phosphate preparations, the measured T m The value is fairly constant: T m1 It only changes between 67.0℃ and 68.3℃, and T m2 Vary between 84°C and 85°C. Variation of the factors tested does not affect these two criteria. Therefore, it is not relevant to analyze the response surface for these two responses.

[0213] Analysis of the histidine program showed that pH had a significant effect: high pH (pH 7) increased the rate of multimerization, as well as the generation of acidic variants. As with histidine, the presence of NaCl tended to increase the rate of multimerization, but limited the appearance of acidic variants. Finally, polysorbate 80 had no significant effect on the responses studied. The response surface is shown in Figure 2. Figure 5 and Figure 6 draw.

[0214] To minimize the monomer rate, it is preferred to use a pH of 6-6.5. At the percentages tested, the addition of polysorbate 80 is undesirable because the presence of the surfactant increases the polymer rate. Salt has no significant effect.

[0215] In order to limit the appearance of acidic variants, it is again necessary to adopt a fairly low pH (6-6.5). As with histidine, the presence of NaCl helps to stabilize this standard. However, the presence of polysorbate 80 has no significant effect.

[0216] The effect of the phosphate buffer factor is very close to the effect of the observed histidine factor (pH 6-6.5, without the presence of polysorbate 80). However, it is important to note that the level of polymer rate and the appearance of acidic variants in phosphate are higher than those in histidine. The minimum and maximum values ​​obtained for 15 histidine and 15 phosphate preparations are shown in Table 3.

[0217] Table 3: Minimum and maximum values ​​obtained for 15 histidine and 15 phosphate preparations

[0218]

[0219] Comprehensive overviews were prepared using specialized software applications (Nemrod and MODDE).

[0220] Conclusions of the screening study:

[0221] (1) It is preferred to formulate antibodies in histidine rather than in phosphate to limit the occurrence of multimers and acidic variants;

[0222] (2) For histidine, the most favorable pH partition is between pH 6 and pH 6.5;

[0223] (3) The presence of polysorbate 80 is not desirable at the percentages tested; this surfactant is not conducive to stabilization (according to the criteria studied);

[0224] (4) In histidine, the presence of NaCl in the formulation is desirable. However, low concentrations (maximum 100 mM) must be advantageous.

[0225] Other factors are considered after screening studies. The stability monitoring of the antibody produced previously in batches reveals a high milky appearance (for some batches, > standard IV). This milky evolution is not relevant (no degradation or aggregation is observed) to the physicochemical properties of the antibody, but seems to be relevant to antibody concentration. In order to limit the milky, it seems that antibody concentration is preferably reduced to 10g / L or even 5g / L, and in the worst case, has completed the pre-allocation study with 20g / L. The obtained results of selected preparation (P001) are shown in Table 4.

[0226] Table 4: Milky results for batch P001

[0227]

[0228] For each of the 4 formulations, no milky white color was observed at T0, but after 1 month of storage at +4°C, the levels increased significantly.

[0229] Although batch P001 was not compliant, these results provide insights into the relevance of these choices for the formulations for the next batch. Therefore, additional testing was performed under stress conditions. Five other formulations with or without NaCl using citric acid or histidine buffers were evaluated under stress at 40°C for 15 days. The presence of NaCl and antibody concentration appear to increase the milky phenomenon. A single formulation stands out due to low levels of milky white: histidine buffer at pH 6. The three citric acid formulations with pH below 6 all have strong milky white. Analytical monitoring of these five stressed formulations showed that histidine buffer at pH 6 with 5g / L protein showed the least effect on charge variant spectra and SEC. Histidine, pH 6, 30mM NaCl formulations with 6% sucrose showed low milky white. After stress, the SEC spectrum also remained acceptable. In order to generate diversity in the formulations tested on the next P batch, sucrose was added. Based on all the results obtained and considerations, the final four formulations presented in Table 5 were selected. The selected formulation, pH 6.0 with 25 mM histidine and 6% sucrose, was deemed to be the best performing formulation for early clinical development.

[0230] Table 5: Final selection of 4 formulations for batch P003

[0231] Preparation code Buffer composition Antibody concentration a 25 mM Histidine, pH 6.0 5g / L b 25 mM histidine, pH 6.0, sucrose 6% 5g / L c 25 mM histidine, pH 6.0, 30 mM NaCl 5g / L d 25 mM histidine, pH 6.5, 100 mM NaCl 5g / L

[0232] Development of highly concentrated formulations

[0233] Additional formulation development studies were performed to achieve a highly concentrated solution of anti-IGF-1R antibody. A formulation containing 25 mM histidine, 5% D-sorbitol, pH 6.0 was selected for future DS preparations.

[0234] The first four preparations of 125mg / mL were developed and appropriate tests were performed. As previously established, the histidine of 25mM concentration was used as pH buffer in the pharmaceutical preparation. Sucrose and D-sorbitol were selected as osmotic pressure regulators (tonicity agents). Polysorbate 80 (PS80) was included as a surfactant in two preparations in four selected preparations. The composition of the selected preparation is presented in Table 6, and the test plan is included in Table 7.

[0235] Table 6: Selected Composition of 125 mg / mL Formulations

[0236]

[0237] Table 7: Testing plan for further development of high-dose formulation

[0238]

[0239] The data generated by the formulation development are summarized below.

[0240] After 12 weeks of duration, the samples kept at -20°C and 2-8°C were taken out and visually evaluated. All preparations remained clear, without milky white, and were light amber. At -20°C and 2-8°C, only very small amounts of visible particles were present in some samples. The SEC-HPLC results obtained after 12 weeks at -20°C are shown in Table 8. All preparations did not show an obvious increase in aggregation, in which case the value of high molecular weight substances (HMWS) was comparable to the value of the initial test at T0. The SEC-HPLC results obtained after storage at 2-8°C for 12 weeks are shown in Table 9. At this temperature, there was no significant difference in stability between the preparations, only a small amount of increase in HMWS was observed, with a difference of about 0.25%. After 2 weeks at 25°C, all preparations showed that monomer % was only slightly reduced, down by about 0.3%, and at this temperature and time point, there was no observable difference in stability between the preparations (Table 10). After 2 weeks at 40°C, the % monomer in all formulations decreased slightly, by about 1.1%, indicating very good stability, and no observable differences in stability between the 4 formulations (Table 11). After 2 weeks at 50°C, the decrease in % monomer in all formulations was more pronounced, with an overall decrease of about 5.3%. Formulations 1b and 3b containing PS80 appeared to have a very slight decrease in stability (Table 12). There were no significant differences between the formulations sucrose and sorbitol.

[0241] Table 8: Thermal stability: -20°C SEC-HPLC results

[0242]

[0243] Table 9: Thermal stability: 2-8°C SEC-HPLC results

[0244]

[0245]

[0246] Table 10: Thermal stability: 25°C SEC-HPLC results

[0247]

[0248] Table 11: Thermal stability: 40°C SEC-HPLC results

[0249]

[0250]

[0251] Table 12: Thermal stability: 50°C SEC-HPLC results

[0252]

[0253] CEX-HPLC analysis revealed that the formulations showed good chemical stability after 12 weeks at -20°C (Table 13) and 2-8°C (Table 14). From the CEX-HPLC data, slightly higher values ​​of the % of major species were observed for the samples kept at 2-8°C. This suggests that the freeze-thaw cycles experienced by the samples at -20°C did not affect aggregation, but were more pronounced on the chemical stability of the molecule.

[0254] Table 13: Thermal stability: -20°C CEX-HPLC results

[0255]

[0256]

[0257] Table 14: Thermal stability: 2-8°C CEX-HPLC results

[0258]

[0259] DLS measurements revealed that after 12 weeks at both temperatures, the particle size Z D PDI values ​​showed an increasing trend over time, but all formulations remained monodisperse after 12 weeks ( Figure 7 and Figure 8 ).

[0260] in conclusion :

[0261] The results obtained lead to the conclusion that formulation 3a without PS80, containing 25 mM histidine and sorbitol as a tonicity modifier, provides slightly better stability than the other formulations. The formulation containing 25 mM histidine and sucrose exhibited a profile similar to that exhibited by the formulation with sorbitol. Therefore, the drug substance formulated with 25 mM histidine, 6% sucrose at pH 6.0, which was initially selected for 20 mg / mL of the drug substance, is considered safe and acceptable for the preparation of a 125 mg / mL clinical product.

[0262] Establishment of the manufacturing process for high-dose antibody drug product (125 mg / mL)

[0263] The drug product preparation process for the low-dose drug product for Phase 1 clinical trial is shown in Fig. 9 The antibody drug substance (20 mg / mL) was thawed at 2-8° C. The formulation was then sterile filtered, filled into vials and sealed.

[0264] In order to concentrate the drug substance to 125mg / mL before manufacturing high-dose drug products, a tangential flow filtration (TFF) step is performed. TFF is also referred to as a cross-flow filtration system, which is a fast and efficient method for concentrating a solution by removing fluid while retaining solute molecules. This process is accomplished by selecting a filter or membrane with a pore size significantly smaller than the solute molecules to allow the retention of solute molecules. The Minimate EVO system of Pall Corporation (Pall) is used in this process, which has a pump, a pressure gauge, a retentate screw clamp, a reservoir, and a pipeline connector.

[0265] The concentration process was developed on a small scale using bulk drug substance from GMP batches as discussed below.

[0266] A PES membrane with a molecular weight cutoff (MWCO) of 30 kDa was used to concentrate the drug product solution from 20 mg / mL to a target concentration of 125 mg / mL. The TFF process flow diagram is presented in Fig.10 middle.

[0267] The process of concentrating the drug product solution begins with the preparation of a formulation buffer containing histidine and sucrose. The formulation buffer is used to precondition the TFF membrane prior to adding the sample. Preconditioning is used to remove air bubbles and equilibrate the system.

[0268] After buffer preconditioning, the thawed drug substance solution (20 mg / mL) was added and mixed using a peristaltic pump at a flow rate of about 40 ml / min. The drug substance solution was concentrated in a container to a final volume of 18-20 mL. After collecting the retentate, the system was rinsed with the formulation buffer added to the concentrated product intermediate. The concentrated product intermediate was filtered through a 0.4 μm prefilter followed by two 0.22 μm PVDF filters. The filtered solution was aliquoted into 2R Class 1 glass vials and stoppered. The process parameters and components used in the preparation of this technical batch are representative of the 125 mg / mL clinical batch proposed for preparation. The technical batch was tested using the same method as that used for the clinical DP batch release, and the technical batch was stabilized as a representative batch to generate stability data before preparing the GMP batch.

[0269] Compatibility in use

[0270] Antibody drug products (DP) are provided as solutions for subcutaneous injection (SC) and intravenous infusion (IV). For administration by intravenous infusion, the antibody is diluted with saline solution, while no dilution is required for subcutaneous administration. In-use compatibility studies have shown that the diluted drug product has good stability in saline at various holding conditions and infusion rates for 2.1 mg / mL and 0.07 mg / mL.

[0271] ● Ambient temperature for up to 24 hours, rapid infusion rate of 100 mL / hour

[0272] ● Ambient temperature for up to 24 hours, slow infusion rate of 50 mL / hour

[0273] Refrigeration for 24 hours, followed by another 24 hours at ambient temperature with a rapid infusion rate of 100 mL / hour

[0274] Refrigeration for 24 hours, followed by another 24 hours at ambient temperature with a slow infusion rate of 50 mL / hour

[0275] In-use compatibility studies confirm that the drug product does not adhere to bags and / or tubing, and there is no aggregation and / or material loss during the filtration phase. The infusion sets used in the in-use compatibility studies are representative of the infusion sets proposed for the clinical studies. Therefore, the antibody is compatible with the selected infusion system. In-use compatibility studies bracket the proposed infusion parameters as defined in the Pharmacy Manual and support the accurate dosing of antibody drug products in the clinic.

[0276] Clear ethylene vinyl acetate (EVA) infusion bags (100 mL) equipped with three connection points (supplied by ICU Medical, P / N SN2010BP) and pump / gravity Clearlink kit (supplied by Baxter International Inc, P / N BXISMMC96901L) with inline BCV, vented chamber with 15 micron filter, ClickLuer, SafePrime, line markers were used in the study. Drug substance batch T102 formulated in histidine and sucrose formulation was tested. T102 DS was identified as the reference standard.

[0277] Two concentrations of the diluted solution in saline solution were tested, 2.1 mg / mL and 0.07 mg / mL. The total volume after dilution was greater than 100 mL to allow the bulk solution to be filled into two infusion bags for each storage condition. A TO sample was removed and analyzed in duplicate for both concentrations under each storage condition.

[0278] For compatibility studies, a 50 mL fill volume was selected, which accounts for 50% of the maximum volume of the infusion bag to simulate a worst-case scenario with a large surface area contact. The lowest concentration in the bag was considered the worst-case scenario due to the high surface to protein exposure ratio. The infusion bags were stored at room temperature and 2-8°C. After the planned storage, the infusion bag was withdrawn and connected to the rest of the infusion set, and the solution was passed through the infusion line / filter at an infusion rate of 100 mL / 1 hour ("fast") and 50 mL / 1 hour ("slow"). After the infusion process was completed, the samples were analyzed. Two samples were extracted for each infusion rate. Fig.11 A representative diagram is provided in order to show the "fast" and "slow" ports to which the other infusion sets described above are connected.

[0279] For infusion bags kept at room temperature, samples were analyzed after two hours (time point (T) = 2 hours), four hours (T = 4 hours), and twenty-four hours (T = 24 hours).

[0280] For infusion bags kept at 2-8°C: keep the bags at 2-8°C for 24 hours. At the end of the storage period, remove the infusion bags from the refrigerator to equilibrate to ambient temperature within 2 hours. Take samples for analysis at the end of the 2-hour equilibration period (T=2 hours), after an additional two hours (T=4 hours), and after twenty-four hours from the time of removal from the refrigerator (T=24 hours). For all samples, test items included appearance, A 280 The protein content obtained by SEC-HPLC and CEX-HPLC was determined. The method was run under the conditions where the system was suitable to ensure the confidence of the results.

[0281] The results of the appearance and protein content in the in-use compatibility studies are presented in Tables 15 to 22. The trends of the monomer content by SEC-HPLC and the major form content by CEX-HPLC are shown in Tables 15 to 22. Figure 12-15 All four quality attributes of the diluted antibody drug product demonstrated excellent stability throughout storage in the infusion bag at both ambient and refrigerated temperatures and after passing through the infusion line and filter at both fast and slow infusion rates.

[0282] Table 15: Results of In-Use Compatibility Study at Room Temperature, 2.1 mg / mL in Saline and an Infusion Rate of 100 mL / 1 Hour

[0283]

[0284] Table 16: Results of In-Use Compatibility Study at Room Temperature, 0.07 mg / mL in Saline and an Infusion Rate of 100 mL / 1 Hour

[0285]

[0286] Table 17: Results of In-Use Compatibility Study at Room Temperature, 2.1 mg / mL in Saline and an Infusion Rate of 50 mL / 1 Hour

[0287]

[0288] Table 18: Results of In-Use Compatibility Study at Room Temperature, 0.07 mg / mL in Saline and an Infusion Rate of 50 mL / 1 Hour

[0289]

[0290] Table 19: Results of the in-use compatibility study at 2-8°C, 2.1 mg / mL in saline and an infusion rate of 100 mL / 1 hour

[0291]

[0292]

[0293] Table 20: Results of the in-use compatibility study at 2-8°C, 0.07 mg / mL in saline and an infusion rate of 100 mL / 1 hour

[0294]

[0295] Table 21: Results of In-Use Compatibility Study at 2-8°C, 2.1 mg / mL in Saline and an Infusion Rate of 50 mL / 1 Hour

[0296]

[0297] Table 22: Results of In-Use Compatibility Study at 2-8°C, 0.07 mg / mL in Saline and an Infusion Rate of 50 mL / 1 Hour

[0298]

[0299] Example 2: High-concentration formulation development and injectability studies

[0300] Study the high concentration behavior of the antibody in the lead buffer selected from the previous stage:

[0301] - Buffer 3a: 25 mM L-histidine, 5% w / v D-sorbitol, pH 6.0.

[0302] - VB421 was concentrated to 150 mg / mL, 175 mg / mL and 200 mg / mL (125 mg / mL was also tested, although stability at this concentration was understood).

[0303] Stability at these concentrations was assessed by visual appearance, SEC, and DLS. Viscosity was also measured and syringeability was assessed to ensure that the injection force was not too high.

[0304] Buffer exchange:

[0305] Loading samples into Sartorius 20 (20 mL capacity, 30 kDa MWCO, PES membrane (DS details: 20 mg / mL, F50085, Hz208F2-4, G101D)). Five cycles of buffer exchange were performed.

[0306] concentration:

[0307] The samples were concentrated to 125 mg / mL, 150 mg / mL, 175 mg / mL and 200 mg / mL. The concentrations were confirmed by protein A280 measurement.

[0308] Observations:

[0309] As before, no problems were encountered during concentration; no visible fiber / particle formation or clogging of the filter membranes.

[0310] Appearance Method

[0311] The vials were gently swirled and observed under an off-white light source (10 W LED, 2000-3750 lux). The vials were observed against both a black background and a white background.

[0312] At elevated concentrations, the only perceptible visual effect was a slightly darker amber color than that observed at 125 mg / mL. All samples were free of opalescence and visible aggregates.

[0313] SEC-HPLC method

[0314] Samples were analyzed at 1 mg / mL (diluted in mobile phase). Injection volume was 10 μL. Buffer was 150 mM sodium phosphate, pH 6.9. Run: Isocratic, 0.35 mL / min, 15 min. Column temperature: Ambient. Column: Agilent AdvanceBio SEC column (p / n 1580-5301). HPLC system: Dionex Ultimate-3000 UHPLC system. Detection wavelength: 280 nm. Samples were injected in duplicate - average results reported.

[0315] There was a small increase in the amount of HMWS (0.2%) when concentrating DS starting from 20 mg / mL. There was no further increase in HMWS between 125 mg / mL and 200 mg / mL, and the monomer % was also similar. For all concentrations, LMWS was about 1.5%.

[0316] Zetasizer Method:

[0317] System: Zetasizer Ultra. Cell: ZEN2112, low volume quartz batch cuvette. Measuring temperature: 25°C. Equilibration time: 120 sec. Data processing: General purpose. Detection angle: Backscatter. Attenuation: Automatic. Measurement process: Automatic. No pause after subrun. No filter. Pause between replicates: 0 sec. 1× replicate; 5× measurements per replicate. Sample preparation: Centrifugation and filtration through a 0.22 μm filter. Samples were analyzed neat, sample volume was approximately 50 μL.

[0318] At higher concentrations, the z-average (Z D ) did not increase significantly. At higher concentrations, the PDI also increased but remained <0.2, indicating a high level of monodispersity. It is interesting to note that the lowest Z was seen for 150 mg / mL. D and PDI value.

[0319] Viscosity measurement

[0320] Viscosity was measured on a RheoSense m-VROC viscometer. Measurements were recorded at 21°C. Rate scans were performed for syringeability assessment by varying the shear rate between 10-90% of the chip's range. The viscosity at 150 mg / mL was slightly lower than that at 125 mg / mL, which was consistent with the DLS results, showing a similar trend.

[0321] Injectability assessment

[0322] Many high concentration therapeutic agents exhibit non-Newtonian (shear thinning) behavior - meaning that viscosity decreases at high shear rates. During injection through a 27G needle, >100,000 s can be achieved at an injection rate of 0.1 ml / s. -1 If syringeability is evaluated solely by low shear rate viscosity measurements, a higher-than-expected injection force may be calculated. This may lead to the removal of a potential formulation of interest from further consideration based on an incorrect syringeability assessment.

[0323] The injection force for non-Newtonian fluids can be calculated as follows:

[0324]

[0325] in:

[0326] F v = injection force (N), l n = needle length (m), σ w = Shear stress at the needle wall (Pa) calculated by plotting shear stress and shear rate. p = Inner radius of the syringe (m). n = Inner radius of the needle (m).

[0327] In calculating the force (F v - in N), this force can be used as an approximate guide to the ease of fluid injection. Recent research (Robinson et al., Adv. Healthcare. Mater. 2020, 9, 1901521) shows the following: v Injection requires minimal effort when F < 12N. v =12-38N, you need to work hard. v = 38-64N, a huge effort is required. v In the case of >64N, injection is not possible.

[0328] This method only estimates the contribution of viscosity to the injection force of the fluid in the syringe. Friction is not taken into account. Friction varies with and depends on the syringe piston and barrel material. This calculation does not take into account the effect of lubrication on the barrel and piston caused by the drug in the syringe. In addition, the resistance to injecting the fluid into the subcutaneous tissue is not taken into account.

[0329] The shear rate ranges from 300-100000s -1 The change results in a viscosity drop of approximately 1 cP. There is a linear relationship between shear rate and shear stress.

[0330] At 125 mg / mL, the viscosity is relatively low (9.5-8.5 cP). The injection forces of various needle / syringe combinations were calculated. The preferred combinations are underlined in Table 23 and produce injection forces that are well within the desired range (<12 N). The outer diameter of the "26s" needle is generally the same as that of a normal 26G needle, but with a smaller inner diameter and thicker walls to improve durability, which makes the injection force of this variant significantly higher.

[0331] Table 23: Syringability evaluation results at 125 mg / ml.

[0332]

[0333]

[0334] The shear rate ranges from 300-100000s -1 The change resulted in a viscosity drop of approximately 1 cP. At 150 mg / mL, the viscosity appeared to be lower. There was a linear relationship between shear rate and shear stress. At 150 mg / mL, non-Newtonian behavior was observed at higher shear rates. As with the 125 mg / mL sample, the only syringe / needle combination that created potential syringability issues was the 26s needle.

[0335] Table 24: Syringability evaluation results at 150 mg / ml.

[0336]

[0337] Increase the shear rate from 250-100000s -1 The change resulted in a viscosity drop of approximately 2.5 cP. There was a linear relationship between shear rate and shear stress. At 175 mg / mL, non-Newtonian behavior was observed at higher shear rates. Again, the only sample that showed potential issues was the 26s needle.

[0338] Table 25: Syringability evaluation results at 175 mg / ml.

[0339]

[0340] Increase the shear rate from 250-100000s -1 The change resulted in a viscosity drop of approximately 2.5 cP. There was a linear relationship between shear rate and shear stress. At 200 mg / mL, non-Newtonian behavior was observed at higher shear rates. Again, the only sample that showed potential issues was the 26s needle.

[0341] Table 26: Syringability evaluation results at 200 mg / ml.

[0342]

[0343]

[0344] Injectability Assessment - Conclusion

[0345] At higher concentrations, more pronounced non-Newtonian behavior was observed. The antibody appears to be injectable under most conditions tested. At 200 mg / mL, the injection force at a rate of 200 cubic millimeters / second is <10 N. The preferred combination of a 3 mL syringe and a 26G needle yields a calculated injection force of 2.5-4.7 N, which is below the recommended value of 12 N, at which effort is required to inject the fluid.

[0346] Concentrating the antibody to 200 mg / mL did not impair visual appearance and remained free of milky white and visible aggregates during the entire concentration step. SEC and DLS did not change significantly between 125 mg / mL and 200 mg / mL. Viscosity did increase, particularly at 175 mg / mL and 200 mg / mL, but the values ​​were still 14-16 cP. The injectability of the antibody showed that at all concentrations, the injection force was <5 N, which indicates that low effort is required to inject this composition.

[0347] Example 3: Deamidation and oxidation analysis

[0348] The samples were analyzed for deamidation and oxidation in Table 27.

[0349] Table 27: Samples used for deamidation and oxidation analysis.

[0350]

[0351] Table 28: Detailed information of formulation samples.

[0352]

[0353]

[0354] Sample preparation: buffer exchange to Tris-HCl pH 8, digestion using immobilized trypsin (Smart Digest, Thermo Scientific), reduction with DTT.

[0355] RP-ESI-MSe analysis: System: Waters NanoAquity UPLC consistent with Xevo G2S QTOF; Injection volume: 2 μL (1 μg IgG); Separation: RP on Waters Acquity UPLC BEH300 C18 1.7 μm 2.1 x 100 mm column. Buffer A: 0.1% FA in water, Buffer B: 0.1% FA in acetonitrile; Elution with linear gradient from 3% to 50% B; Mass spectrometry: positive ion mode, MSe scan: 250-2000 m / z.

[0356] For UNIFI TM and PTM identification criteria for further manual investigation: accurate mass of intact peptide within 20 ppm, fragment ion pattern used to confirm identity, and isotope pattern clearly resolved.

[0357] Only deamidation was identified in HC. Between N and Q, deamidation was identified only at N, and most fragment ions containing N or Q were not found to be deamidated. No second deamidation (2x) was found on any deamidated peptide in both HC and LC.

[0358] Table 29: List of fragments of antibodies and their deamidation.

[0359]

[0360]

[0361] Only deamidation was identified at HC. Between N and Q, deamidation was identified only in N, and most fragment ions containing N or Q were not found to be deamidated. At HC:T35 (2x), the N residue of the peptide was found to be deamidated (the second deamidation is indicated as 2x).

[0362] Oxidation was identified at both the M and W residues of the fragment ions, but most fragment ions containing both the M and W residues were not oxidized. Under HC:T24, two W residues of the peptide chain were found to be oxidized (the second oxidation was indicated as 2x). Under LC:T1, 1x oxidation was also observed.

[0363] Table 30: List of fragments of antibodies and their oxidation.

[0364]

[0365] Oxidation was identified at both the M and W residues of the fragment ion. Under HC:T24, two W residues of the peptide chain were found to be oxidized (the second oxidation is indicated as 2x). Under LC, no oxidation was observed.

[0366] PTMs of VAIO-1799 samples were analyzed using deamidation on Q and N and oxidation at M and W. Peptides were resolved by reverse phase LC-MS and identified by accurate mass and fragment sequence tags. However, deamidation was identified at N and oxidation was identified at both M and W. Respectively, the deamidation observed in 1a and 3a increased over the deamidation observed in 1b and 3b. The oxidation observed in 1b increased over the oxidation observed in 1a (especially on HC-88-124). The oxidation observed in 3a also increased similarly over the oxidation observed in 3b.

[0367] Example 4: Safety, Tolerability and Pharmacokinetics

[0368] A Phase I trial was conducted to evaluate the safety, tolerability, pharmacokinetics, and pharmacodynamics (PD) of single ascending doses (SAD) of intravenous or subcutaneous ronituzumab in healthy participants. The study conducted was a single-center, randomized, double-blind, placebo-controlled, sequential single ascending dose (SAD) study. The study involved eight (8) healthy volunteer groups divided into two groups: four (4) groups received intravenous administration of ronituzumab, and four (4) groups received subcutaneous administration of ronituzumab.

[0369] Specifically, groups 1-4 received intravenous administration of 0.1 mg / kg, 0.3 mg / kg, 1.0 mg / kg or 3.0 mg / kg of ronituzumab or matching placebo over the course of 60 minutes. Groups 5-8 received a fixed-dose subcutaneous injection dose of 20 mg, 40 mg, 125 mg or 250 mg of ronituzumab or matching placebo administered in a volume of 2 mL or less.

[0370] According to the study protocol, the participants in the study were healthy. Specifically, the participants were between 18 and 55 years old and had a body mass index (BMI) of 18-32 kg / m at the time of their inclusion in the study. 2 in the range between.

[0371] During the study, routine laboratory, audiological, pharmacokinetic, and pharmacodynamic assessments were performed at pre-determined time points in each cohort participating in the study. In addition, the safety and tolerability of the administered medications were evaluated throughout the study by the assessment of adverse events and were monitored for 99 days.

[0372] Pharmacokinetic data were obtained using a validated electrochemiluminescent immunoassay. Receptor occupancy was assessed using a qualified flow cytometric assay.

[0373] The study involved the inclusion of sixty-three (63) participants. Of these participants, forty-seven (47) received ronitumumab and sixteen (16) received placebo. Four (4) participants discontinued from this study due to loss to follow-up or other reasons. This loss of participants was not considered to affect the primary analysis.

[0374] Baseline characteristics and demographics of the participants are provided in Table 31 below.

[0375] Table 31: Baseline characteristics:

[0376] Safety group (n=63) Age (years) 35.7(19-55) gender male 48(76.2) female 15(23.8) Race American Indian or Alaska Native 1(1.6) Black / African American 34(54) White 23(36.5) unknown 3(4.8) Multi-ethnic 2(3.2) Weight(kg) 78.2(52.9-100.1) <![CDATA[BMI(kg / m 2 )]]> 25.67(20.0-29.6)

[0377] The data in Table 31 are presented as n (%) or mean (range). There were no meaningful differences in any characteristic across treatment groups.

[0378] Preliminary data evaluating safety and tolerability by tracking treatment-emergent adverse events (TEAEs) are presented in Fig.16 middle.

[0379] The data showed that no serious adverse events (SAEs) occurred. All adverse events were mild and moderate in severity, and dose-related safety events occurred. Overall, closely monitored target-related adverse events of special interest (AESI) included type I allergic reactions / anaphylaxis, hyperglycemia, infusion-related reactions, diarrhea / inflammatory bowel disease exacerbation, hearing impairment, and muscle spasms. In this study, one (1.6%) AESI was observed in one (1) participant; gastrointestinal disorders (abdominal pain, bloating, and frequent bowel movements) that were "possibly" or "probably" related to lonizumab were observed in the 20 mg SC group (participants reported more than 1 TEAE).

[0380] One participant had an infusion-related reaction, and three (3) had injection site reactions that were “possibly” or “probably” related to ronitumumab. All reactions were mild and self-limited, resolving within a few hours without medical intervention.

[0381] Pharmacokinetic Assessment:

[0382] The mean serum ronitumumab concentration profile over time after intravenous (IV) administration is presented in Fig.17 The mean serum lonizumab concentration profile over time after subcutaneous (SC) administration is presented in Fig.18 middle.

[0383] exist Fig.17 and 18 In the data presented in , samples below the limit of quantification (0.1 μg / mL) are plotted as 0 μg / mL in 3.0 mg / kg, and n does not include 1 participant who discontinued early in the study due to deployment as mentioned previously. Fig.17 and Fig.18 The shaded area in the graph represents serum concentrations of ronitumumab that were above saturation of IGF-1R internalization.

[0384] As from Fig.17 and Fig.18 As is evident from the data presented in , mean serum concentrations of ronitumumab increased in a greater than dose-proportional manner across the dose range tested. Fig.17 and Fig.18Also provided is an indication of target-mediated drug disposition (TMDD) after intravenous and / or subcutaneous administration of ronitumumab. TMDD describes a nonlinear pharmacokinetic (PK) phenomenon caused by the high affinity binding of a compound to its pharmacological target. After ronitumumab binds to IGF-1R, the resulting complex will degrade. At low concentrations, the serum concentration of ronitumumab decreases rapidly due to the efficient binding and degradation of ronitumab to IGF-1R, and thus, the measured serum concentration of ronitumumab decreases rapidly. On the contrary, at high concentrations, the target IGF-1R is saturated by ronitumumab, and the serum concentration of ronitumumab decreases slowly. This slow decline in the serum concentration of ronitumumab indicates that it can be used to bind to the newly generated IGF-1R. In addition to the pharmacology of ronitumumab, this data also provides evidence of additional IGF-1R internalization.

[0385] This data suggests that the TMDD threshold for achieving the lower limit of saturation of IGF-1R internalization appears to be about 3 μg / mL ronitumumab. Compared to intravenous administration, subcutaneous administration of ronitumumab overcomes the TMDD to maintain pharmacologically relevant concentrations of ronitumumab.

[0386] Pharmacodynamic evaluation

[0387] Mean ronituzumab receptor occupancy over time on peripheral blood mononuclear cells after intravenous administration is provided in Fig.19 Mean lonizumab receptor occupancy over time on peripheral blood mononuclear cells after subcutaneous administration is provided in Fig. 20 middle. Fig.19 and Fig. 20 The shaded area in the Figure indicates the region of optimal IGF-1R internalization following administration of the pharmaceutical composition comprising ronitumumab.

[0388] like Fig.19 and 20 As mentioned in the data provided in, in all groups, maximum IGF-1R occupancy was observed by the first time point (12 hours). The duration of receptor saturation increased with increasing doses of ronitumumab. 1.0 mg / kg and 3.0 mg / kg (administered as intravenous infusion) and 125 mg and 250 mg (administered as subcutaneous injection) maintained at maximum receptor occupancy levels for a period of at least 4 weeks. As previously mentioned, in the 3.0 mg / kg group, n does not include one (1) participant who discontinued early in the study. Fig.19 and 20 The shaded area in the graph indicates receptor saturation.

[0389] Example 5: Multiple-dose trial for subcutaneous administration:

[0390] In the planned trial, 36 participants with thyroid eye disease (TED) will be enrolled in a multicenter, double-blind, placebo-controlled, randomized, multiple ascending dose (MAD) clinical study to evaluate ronitumumab relative to placebo. This study will involve up to 3 cohorts, with each cohort receiving three (3) different treatment groups.

[0391] The proposed eligibility criteria for participation in this study are provided below:

[0392] Eligible age for study: 18 to 65 years (adults, older adults)

[0393] Eligible gender for the study: All

[0394] standard

[0395] Key inclusion criteria:

[0396] ●Male or female, aged ≥18 years and ≤65 years.

[0397] ● Proptosis was defined as ≥3 mm above normal in the study eye.

[0398] Clinical Activity Score (CAS) ≥4 (using a 7-item scale) in the most affected eye

[0399] Active TED symptoms within 24 months prior to baseline

[0400] ●Must agree to use highly effective contraception as specified in the protocol

[0401] Key exclusion criteria:

[0402] Biopsy-proven or clinically suspected inflammatory bowel disease or irritable bowel syndrome.

[0403] ●Clinically significant pathological blood tests related to hearing

[0404] Corneal decompensation unresponsive to medical management.

[0405] ● Previous orbital irradiation (for any reason) or any previous surgical treatment for TED.

[0406] ●Subjects with diabetes or hemoglobin A1c>6.0%.

[0407] ●Use of any steroid (intravenous [IV] or oral) equivalent to a cumulative dose of >3 g methylprednisolone for the treatment of TED.

[0408] Prior use of steroids (intravenous or oral) specifically for the treatment of TED, with a total dose not exceeding 1 g in the 8 weeks prior to screening.

[0409] ●Prior use of tefixime or any other IGF-1 receptor (IGF-1R) inhibitor.

[0410] Any prior treatment of TED with biologic drugs (eg, rituximab and tocilizumab).

[0411] Cohort 1 will receive a single subcutaneous injection of 20 mg ronitumumab or placebo on Days 1 and 21.

[0412] Cohort 2 will receive a single subcutaneous injection of 125 mg ronitumumab or placebo on Days 1 and 21.

[0413] Cohort 3 will receive a single subcutaneous injection of 250 mg ronitumumab or placebo on Days 1 and 21.

[0414] The primary outcomes to be measured in the study will include the incidence and characterization of non-serious treatment-emergent adverse events (TEAEs) occurring between Day 1 and Day 113. The outcomes measured will also include the incidence and characterization of serious TEAEs occurring between Day 1 and Day 113.

[0415] Secondary outcomes measured will include:

[0416] 1. PK profile of ronitumumab [time frame: day 1 to day 113]

[0417] the area under the concentration-time curve from time zero to infinity (AUCO-infinity);

[0418] 2. PK profile of ronitumumab [time frame: day 1 to day 113]

[0419] Maximum observed concentration (Cmax)

[0420] 3. PK profile of ronitumumab [time frame: day 1 to day 113]

[0421] The time when C is maximum (Tmax)

[0422] 4. PK profile of ronitumumab [time frame: day 1 to day 113]

[0423] The area under the concentration-time curve (AUC) from time zero to the last quantifiable concentration O -at last)

[0424] 5. PK profile of ronitumumab [time frame: day 1 to day 113]

[0425] Elimination half-life (T1 / 2 elimination)

[0426] 6. PK profile of ronitumumab [time frame: day 1 to day 113]

[0427] Elimination rate constant (Kel)

[0428] 7. PK profile of ronitumumab [time frame: day 1 to day 113]

[0429] Total body clearance (CL / F)

[0430] 8. PK profile of ronitumumab [Time frame: Day 1 to Day 113]

[0431] Volume of distribution (Vz / F)

[0432] 9. Proportion of subjects who developed anti-drug antibodies (ADA) after administration of multiple doses of lonizumab [Time frame: Day 1 to Day 113]

[0433] Pharmacokinetic and pharmacodynamic data

[0434] Incorporated by Reference

[0435] Other documents, such as patents, patent applications, patent publications, journals, books, papers, web page content, publicly available databases, have been referenced and cited throughout this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes.

[0436] Equivalent form

[0437] Various modifications to the invention and many other embodiments of the invention in addition to the embodiments shown and described herein will become apparent to those skilled in the art based on the entire content of this document (including references to the scientific literature and patent literature cited herein). The subject matter of this article contains important information, illustrations, and guidance that can be adapted to practice the invention in its various embodiments and equivalent forms.

Claims

1. A pharmaceutical composition comprising: (a) at least 75 mg / ml of an anti-IGF-1R antibody comprising a CDRH1 of SEQ ID NO: 1, a CDRH2 of SEQ ID NO: 2, a CDRH3 of SEQ ID NO: 3, a CDRL1 of SEQ ID NO: 4, a CDRL2 of SEQ ID NO: 5, and a CDRL3 of SEQ ID NO: 6; (b) 20 mM to 30 mM histidine; and (c) 4% to 6% D-sorbitol; The pH of the pharmaceutical composition is 5.5-6.

5.

2. The pharmaceutical composition according to claim 1, wherein the antibody comprises a heavy chain variable domain, and the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:

7.

3. The pharmaceutical composition according to claim 1 or 2, wherein the antibody comprises a light chain variable domain, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:

8.

4. The pharmaceutical composition of claim 1, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12 or SEQ ID NO:

13. 5 . The pharmaceutical composition according to claim 1 or 4 , wherein the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:

10. 6 . The pharmaceutical composition according to any one of claims 1 to 3 , wherein the antibody comprises a human IgG1 heavy chain constant domain and a human kappa light chain constant domain.

7. The pharmaceutical composition according to claim 1, wherein the antibody is lonigutamab.

8. The pharmaceutical composition of any one of claims 1 to 7, wherein the pharmaceutical composition comprises at least 100 mg / ml, at least 125 mg / ml, at least 150 mg / ml, at least 175 mg / ml, at least 200 mg / ml or at least 250 mg / ml of the anti-IGF-1R antibody.

9. The pharmaceutical composition according to any one of claims 1 to 7, wherein the pharmaceutical composition comprises 75 mg / ml to 300 mg / ml, 100 mg / ml to 300 mg / ml or 125 mg / ml to 250 mg / ml of the anti-IGF-1R antibody.

10. The pharmaceutical composition of any one of claims 1 to 7, wherein the pharmaceutical composition comprises about 125 mg / ml, about 150 mg / ml, about 175 mg / ml, about 200 mg / ml or about 250 mg / ml of the anti-IGF-1R antibody.

11. The pharmaceutical composition of any one of claims 1 to 10, wherein the pharmaceutical composition comprises about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM or about 30 mM histidine.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the pharmaceutical composition comprises about 4%, 5% or 6% D-sorbitol.

13. The pharmaceutical composition of any one of claims 1 to 12, wherein the pH of the pharmaceutical composition is about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, or about 6.

5.

14. The pharmaceutical composition according to any one of claims 1 to 13, wherein the osmotic pressure of the pharmaceutical composition is within the physiological osmotic pressure range of 250-400 mOsm / kg.

15. The pharmaceutical composition according to any one of claims 1 to 14, wherein the viscosity of the pharmaceutical composition does not exceed 30 cP at 21°C.

16. The pharmaceutical composition according to claim 15, wherein the viscosity of the pharmaceutical composition does not exceed 15 cP at 21°C.

17. The pharmaceutical composition of claim 15, wherein the viscosity of the pharmaceutical composition is about 10 cP, about 11 cP, about 12 cP, about 13 cP, about 14 cP, about 15 cP, about 16 cP, about 17 cP, about 18 cP, about 19 cP, about 20 cP, about 21 cP, about 22 cP, about 23 cP, about 24 cP, about 25 cP, about 26 cP, about 27 cP, about 28 cP, about 29 cP or about 30 cP at 21°C.

18. A pharmaceutical composition according to any one of claims 1 to 17, wherein the pharmaceutical composition is stable for at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, at least 13 weeks, at least 14 weeks, at least 15 weeks or at least 16 weeks.

19. The pharmaceutical composition according to claim 18, wherein the pharmaceutical composition is stable at a temperature of -70°C to 40°C.

20. A method of treating thyroid eye disease (TED), the method comprising administering a pharmaceutical composition according to any one of claims 1 to 19.

21. The method of claim 20, wherein the pharmaceutical composition is administered subcutaneously.

22. The method of claim 20, wherein the pharmaceutical composition is administered intramuscularly.

23. The method of any one of claims 20 to 22, wherein the pharmaceutical composition is administered in a delivery volume of no more than 2 ml.

24. The method of any one of claims 20 to 23, wherein the pharmaceutical composition is administered via a needle no larger than 24G in size.

25. The method according to any one of claims 20 to 24, wherein the pharmaceutical composition is administered with an injection force not exceeding 12N.

26. The method of claim 25, wherein the pharmaceutical composition is administered with an injection force of about 4N, about 5N, about 6N, about 7N, about 8N, about 9N, about 10N, about 11N, or about 12N.

27. The method of any one of claims 20 to 26, wherein the method reduces the severity of the thyroid eye disease (TED).

28. The method of any one of claims 20 to 27, wherein the method reduces proptosis in an eye of a subject suffering from thyroid eye disease (TED).

29. The method of claim 28, wherein proptosis is reduced by at least 2 mm.

30. The method of claim 28, wherein proptosis is reduced by at least 3 mm.

31. The method of claim 28, wherein proptosis is reduced by at least 4 mm.

32. The method of any one of claims 20 to 31, wherein the method reduces the Clinical Activity Score (CAS) of Thyroid Eye Disease (TED).

33. The method of claim 32, wherein the Clinical Activity Score (CAS) is reduced by at least 2 points.

34. The method of claim 32, wherein the Clinical Activity Score (CAS) is reduced to one (1).

35. The method of claim 32, wherein the subject's Clinical Activity Score (CAS) decreases to zero (0).

36. The method of any one of claims 20 to 35, wherein the method improves the subject's quality of life.

37. The method of claim 36, wherein the quality of life is measured by the Graves' Ophthalmopathy Quality of Life (GO-QoL) assessment.

38. The method of claim 36, wherein the quality of life is measured by its visual function or appearance subscale.

39. The method of claim 36, wherein the quality of life is measured by the European Group on Graves' orbitopathy (EUGOGO) guidelines.

40. The method of any one of claims 20 to 39, wherein the method reduces the severity of diplopia.

41. The method of claim 40, wherein the diplopia is persistent diplopia.

42. The method of claim 40, wherein the diplopia is non-persistent diplopia.

43. The method of claim 40, wherein the diplopia is intermittent diplopia.

44. A syringe comprising the pharmaceutical composition according to any one of claims 1 to 19.

45. The syringe of claim 44, wherein the syringe comprises a delivery volume of no more than 2 ml.

46. ​​The syringe of claim 44 or 45, wherein the syringe comprises a needle of size no greater than 24G.

47. The syringe according to any one of claims 44 to 46, wherein the syringe is an automated reusable fixed dose pen.

48. The syringe of any one of claims 44 to 46, wherein the syringe is an automated reusable variable dose pen.

49. The syringe according to any one of claims 44 to 46, wherein the syringe is an automated disposable fixed dose auto-injector.

50. A method of treating thyroid eye disease (TED), the method comprising administering the pharmaceutical composition using a syringe according to any one of claims 44 to 49.

51. The method of claim 50, wherein the pharmaceutical composition is administered subcutaneously.

52. The method of claim 50, wherein the pharmaceutical composition is administered intramuscularly.

53. The method of any one of claims 50 to 52, wherein the pharmaceutical composition is administered with an injection force of no more than 12N.

54. The method of claim 53, wherein the pharmaceutical composition is administered with an injection force of about 4N, about 5N, about 6N, about 7N, about 8N, about 9N, about 10N, about 11N, or about 12N.

55. A pharmaceutical composition according to any one of claims 1 to 19 for use in the treatment of thyroid eye disease (TED).

56. A pharmaceutical composition for use according to claim 55, wherein the pharmaceutical composition is administered subcutaneously.

57. The pharmaceutical composition for use according to claim 55, wherein the pharmaceutical composition is administered intramuscularly.

58. A pharmaceutical composition for use according to any one of claims 55 to 57, wherein the pharmaceutical composition is administered in a delivery volume of no more than 2 mL.

59. A pharmaceutical composition for use according to any one of claims 55 to 58, wherein the pharmaceutical composition is administered via a needle of size no greater than 24G.

60. A pharmaceutical composition for use according to any one of claims 55 to 59, wherein the pharmaceutical composition is administered with an injection force of no more than 12N.

61. The pharmaceutical composition for use of claim 60, wherein the pharmaceutical composition is administered with an injection force of about 4N, about 5N, about 6N, about 7N, about 8N, about 9N, about 10N, about 11N, or about 1N.

62. An anti-IGF-1R antibody comprising: (a) a heavy chain comprising CDRH1 of SEQ ID NO: 1, CDRH2 of SEQ ID NO: 2, CDRH3 of SEQ ID NO: 3, and a charged amino acid located at the c-terminus thereof; and (b) a light chain comprising CDRL1 of SEQ ID NO:4, CDRL2 of SEQ ID NO:5, and CDRL3 of SEQ ID NO:

6.

63. The anti-IGF-1R antibody of claim 62, wherein the heavy chain comprises a variable domain comprising the amino acid sequence of SEQ ID NO:

7.

64. The anti-IGF-1R antibody of claim 62 or 63, wherein the light chain comprises a variable domain comprising the amino acid sequence of SEQ ID NO:

8.

65. The anti-IGF-1R antibody of any one of claims 62 to 64, wherein the charged amino acid is a positively charged amino acid.

66. The anti-IGF-1R antibody of claim 65, wherein the positively charged amino acid is lysine, histidine or arginine.

67. The anti-IGF-1R antibody of claim 66, wherein the positively charged amino acid is lysine.

68. The anti-IGF-1R antibody of claim 65, wherein the heavy chain comprises the sequence of SEQ ID NO:

11.

69. The anti-IGF-1R antibody of claim 65, wherein the heavy chain comprises the sequence of SEQ ID NO:

12.

70. The anti-IGF-1R antibody of claim 65, wherein the heavy chain comprises the sequence of SEQ ID NO:

13.

71. The anti-IGF-1R antibody of any one of claims 62 to 64, wherein the charged amino acid is a negatively charged amino acid.

72. The anti-IGF-1R antibody of claim 71, wherein the positively charged amino acid is aspartic acid or glutamic acid.

73. The anti-IGF-1R antibody of claim 71, wherein the heavy chain comprises the sequence of SEQ ID NO:

14.

74. The anti-IGF-1R antibody of claim 71, wherein the heavy chain comprises the sequence of SEQ ID NO:

15.

75. A pharmaceutical composition comprising the antibody according to any one of claims 62 to 74.

76. A method of treating thyroid eye disease (TED), the method comprising administering the pharmaceutical composition of claim 75.

77. The method of claim 76, wherein the pharmaceutical composition is administered subcutaneously.

78. The method of claim 76, wherein the pharmaceutical composition is administered intramuscularly.

79. The pharmaceutical composition according to claim 75, for use in the treatment of thyroid eye disease (TED).

80. The pharmaceutical composition for use of claim 79, wherein the pharmaceutical composition is administered subcutaneously.

81. The pharmaceutical composition for use of claim 79, wherein the pharmaceutical composition is administered intramuscularly.

82. A method of treating thyroid eye disease (TED), the method comprising administering to a patient a pharmaceutical composition comprising a therapeutically effective dose of ronitumumab in the form of an injection.

83. The method of claim 82, wherein the injection is an intravenous infusion.

84. The method of claim 82, wherein the injection is administered subcutaneously.

85. The method of claim 83, wherein ronitumumab is administered as an intravenous infusion at a dose of about 0.1 mg / kg.

86. The method of claim 83, wherein ronitumumab is administered as an intravenous infusion at a dose of about 0.3 mg / kg.

87. The method of claim 83, wherein ronitumumab is administered as an intravenous infusion at a dose of about 1.0 mg / kg.

88. The method of claim 83, wherein ronitumumab is administered as an intravenous infusion at a dose of about 3.0 mg / kg.

89. The method of claim 84, wherein ronitumumab is administered as a subcutaneous injection comprising 20 mg ronitumumab.

90. The method of claim 84, wherein ronitumumab is administered as a subcutaneous injection comprising 40 mg ronitumumab.

91. The method of claim 84, wherein ronitumumab is administered as a subcutaneous injection comprising 125 mg ronitumumab.

92. The method of claim 84, wherein ronitumumab is administered as a subcutaneous injection comprising 250 mg ronitumumab.

93. The method of claim 82, wherein the administration of a therapeutically effective dose of ronitumumab produces a serum concentration of about 3 μg / mL or greater.

94. The method of claim 83, wherein the intravenous administration is performed for a duration of up to about 60 minutes.

95. The method of claim 82, wherein the administration of ronitumumab has minimal side effects.

96. The method of claim 82, wherein the administering of the pharmaceutical composition comprising ronitumumab achieves an IGF-1R occupancy of 95% or greater.

97. The method of claim 96, wherein the 95% or greater IGF-IR occupancy is achievable 1 hour after administration of the pharmaceutical composition.

98. The method of claim 96, wherein after administration of the pharmaceutical composition comprising ronitumumab, an IGF-1R occupancy of 90% or greater is maintained for a duration of at least 28 days.

99. A method of treating thyroid eye disease (TED), the method comprising subcutaneously administering a pharmaceutical composition comprising about 10 mg to about 300 mg of ronitumumab.

100. The method of claim 99, wherein the pharmaceutical composition comprises about 20 mg of ronitumumab.

101. The method of claim 99, wherein the pharmaceutical composition comprises about 125 mg of ronitumumab.

102. The method of claim 99, wherein the pharmaceutical composition comprises about 250 mg of ronitumumab.

103. The method of claim 99, wherein the pharmaceutical composition is administered once a week.

104. The method of claim 99, wherein the pharmaceutical composition is administered once every two weeks.

105. The method of claim 99, wherein the pharmaceutical composition is administered once every three weeks.

106. The method of claim 99, wherein the pharmaceutical composition is administered once every four (4) weeks.

107. The method of claim 100, wherein the pharmaceutical composition is administered on day 1 and day 21.

108. The method of claim 101, wherein the pharmaceutical composition is administered on day 1 and day 21.

109. The method of claim 102, wherein the pharmaceutical composition is administered on day 1 and day 21.

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